Metal complexes derivatized with folate for use in diagnostic and therapeutic applications

ABSTRACT

Diagnostic and therapeutic compositions in the form of complexes for enhancing transmembrane transport of a diagnostic or therapeutic agent and methods for their use. The complexes contain the α, γ, or bis isomers of folate receptor-binding analogs of folate, a metal chelated by a ligand, and in one embodiment, a chemotherapeutic agent.

This application is a divisional of co-pending application Ser. No.09/477,072 filed Jan. 3, 2000, now U.S. Pat. No. 6,221,334 which in turnis a divisional of application Ser. No. 09/080,157 filed on May 16,1998, now U.S. Pat. No. 6,093,382.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to diagnostic and therapeuticcompositions, methods of their use, and processes of their preparation.

More particularly, the invention relates to:

-   -   (a) Magnetic resonance diagnostic compositions for visualization        of tissues that over-express folate binding protein, comprising        ligands chelated to superparamagnetic or paramagnetic metals and        coupled to folate-receptor binding ligands;    -   (b) Radiodiagnostic compositions for visualization of tissues,        comprising ligands chelated to radioactive gamma-emitting metals        and coupled to folate-receptor binding ligands;    -   (c) Compositions for radiotherapy or for neutron capture        therapy, comprising ligands chelated to radioactive alpha or        beta-emitting metals or to metals suitable for neutron capture        therapy and coupled to folate-receptor binding ligands; and    -   (d) Compositions for chemotherapy, comprising certain        derivatives of folic acid coupled to a cancer chemotherapy drug        through the alpha carboxylate of folic acid or coupled through        both the alpha and gamma carboxylates.

2. Reported Developments

The folate-based diagnostic and therapeutic agents of the presentapplication are designed for use in Nuclear Medicine, Magnetic ResonanceImaging (MRI), and neutron capture therapy applications. Magneticresonance (hereinafter sometimes referred to as MR) imaging is wellknown and widely used by the prior art for obtaining spatial images ofparts of a patient for clinical diagnosis. The image is obtained byplacing the patient in a strong external magnetic field and observingthe effect of this field on the magnetic properties of protons containedin and surrounding the organ or tissue of the patient. The protonrelaxation times, called T₁ or spin-lattice or longitudinal relaxationtime, and T₂ or spin-spin or transverse relaxation time depend on thechemical and physical environment of the organ or tissue being imaged.In order to improve the clarity of the image, a diagnostic agent isadministered intravenously (hereinafter sometimes referred to as I.V.)and is taken up by the organs, such as the liver, spleen, and lymphnodes to enhance the contrast between healthy and diseased tissues.

The contrast agents used in MR imaging derive their signal-enhancingeffect from the inclusion of a material exhibiting paramagnetic,ferrimagnetic, ferromagnetic or superparamagnetic behavior. Thesematerials affect the characteristic relaxation times of the imagingnuclei in the body regions into which they distribute causing anincrease or decrease in MR signal intensity. There is a need forcontrast agents such as those of the present invention, that selectivelyenhance signal intensity in particular tissue types, as most MR contrastagents are relatively non-specific in their distribution.

Nuclear medicine procedures and treatments are based on internallydistributed radioactive materials, such as radiopharmaceuticals orradionuclides, which emit electromagnetic radiations as gamma rays orphotons. Following I.V., oral or inhalation administration, the gammarays are readily detected and quantified within the body usinginstrumentation such as scintillation and gamma cameras. Thegamma-emitting agents of the present invention are designed toselectively localize in particular targeted tissues by transmembranetransport, yielding either high signal intensity in these tissue typesfor imaging purposes, or high radiation dose, for radiotherapy purposes.

Transmembrane transport of exogenous molecules, such as diagnosticagents, is also known by the prior art. One method of transmembranedelivery, receptor-mediated endocytosis, is the movement ofextracellular ligands bound to cell surface receptors into the interiorof the cells through invagination of the membrane. This process isinitiated by the binding of a ligand to its specific receptor. Folates,which are required for the survival and growth of eukaryotic cells, aretaken up into cells by receptor-mediated transport after binding tofolate binding protein on the cell membrane. The cellular uptake ofexogenous molecules can be enhanced by conjugation of these molecules tofolate. Such conjugates have been used to target folate receptors toenhance cellular uptake of exogenous molecules, including somediagnostic agents. The uptake of substances by receptor-mediatedendocytosis (hereinafter sometimes termed RME) is a characteristicability of some normal, healthy cells. RME transport systems have beenfound on normal macrophages, hepatocytes, fibroblasts and reticulocytes.On the other hand, conversion of normal cells into tumor cells can beassociated with an increase or decrease in the activity of receptorsperforming RME or, sometimes, with changes in the levels of receptorexpression.

The use of neutron capture therapy for the treatment of cancer is wellknown to those skilled in the art. Briefly the system comprisesadministering a target substance that emits short-range radiation whenit is irradiated with neutrons. Boron-10 has traditionally been used forneutron capture therapy, but more recently Gadolinium-157, which has avery high cross section for neutrons and emits short rangeAuger-electrons, has been used. [Brugger, R. M. and Shih, J. A.,Strahlentherapie Und Onkologie, 165, 153-156, 1989; Brugger, R. M. andShih, J. A., Medical Physics, 19, 733-744, 1992]. Specificity isachieved by using neutrons of appropriate energy and the selectivedistribution of the gadolinium within the tumor tissue. In the past,neutron capture therapy has suffered from insufficient concentration oftarget substance in the desired cells and in the case of gadolinium, hassuffered from the exclusion of the gadolinium from the inside of thecell. The use of the folate-containing gadolinium compounds of thisinvention is advantageous because of the large amounts of gadoliniumthat are specifically taken up by the desired cells. The internalizationof the compounds of this invention following binding to folate bindingprotein is beneficial because of the short range of the Auger electrons.In addition, the gadolinium compounds of this invention can be used asMRI contrast agents that selectively target the cells that are to betreated by neutron capture therapy. The imaging data can provide theradiotherapist with spatial information beneficial for planning theradiotherapy procedure, using the same gadolinium atoms as are used asthe target for the neutrons.

The following illustrative studies describe relevant properties of thefolate receptor.

Folic acid or pteroyl glutamic acid is a vitamin consisting of apteridine ring linked by a methylene bridge to a para-aminobenzoic acidmoiety, which is joined through an amide linkage to a glutamic acidresidue. Folic acid and folates are well absorbed from the dietprimarily via the proximal portion of the small intestine. Followingtheir absorption from the digestive system, dietary folates are rapidlyreduced by dihydrofolate reductase and other enzymes to tetrahydrofolicacid and derivatives thereof.

Folates are required for the survival and growth of eukaryotic cells, sotheir cellular uptake is assured by at least two independent transportmechanisms. Reduced folates are internalized via a carrier-mediated lowaffinity (K_(m) 1-5 μM) anion-transport system that is found in nearlyall cells. Folic acid and 5-methyl tetrahydrofolate can also enter cellsvia a high affinity (K_(d) values in the nanomolar range) membrane-boundfolate-binding protein (hereinafter sometimes referred to as FBP) thatis anchored to the cell membrane via a glycosylphosphatidylinositol(hereinafter sometimes referred to as GPI) moiety. This process has beenstudied in MA104 cells, where experiments have shown that5-methyltetrahydrofolate is taken up into the cell after binding toglycosylphosphatidylinositol (GPI)-anchored FBP that has clustered incell structures known as caveolae. The caveolae then seal the folatebinding protein-folate complex off from the extracellular space andtransport folate into the cell. Once inside, the folate dissociates fromFBP and diffuses into the cytoplasm, where it is rapidly coupled to oneor more glutamic acid residue, slowing diffusion out of the cell. Thecaveolae and FBP then migrate to the membrane surface for another roundof folate uptake.

There are two major isoforms of the human membrane folate bindingproteins, α and β. The two isoforms have ˜70% amino acid sequencehomology, and differ dramatically in their stereospecificity for somefolates. Both isoforms are expressed in both fetal and adult tissue;normal tissue generally expresses low to moderate amounts of FR-β. FR-αis expressed in normal epithelial cells and is frequently strikinglyelevated in a variety of carcinomas, with the exception of squamous cellcarcinomas of the head and neck. Several papers have reported theoverexpression of folate binding protein in cancer. See for example:

-   Ross J F, Chaudhuri P K, Ratman M, “Differential regulation of    folate receptor isoforms in normal and malignant tissues in vivo and    in established cell lines. Physiologic and clinical implications”,    Cancer, 1994, 73(9), 2432-2443;-   Rettig, W, Garin-Chesa P, Beresford H, Oettgen H, Melamed M. Old L.,    “Cell-surface glycoproteins of human sarcomas: differential    expression in normal and malignant tissues and cultured cells”,    Proc. Natl. Acad. Sci U.S.A., 1988, 85, 3110-3114;-   Campbell I G, Jones T A, Foulkes W D, Trowsdale J., “Folate-binding    protein is a marker for ovarian cancer”, Cancer Res., 1991, 51,    5329-5338;-   Coney L R, Tomassetti A, Carayannopoulos L, Frasca V, Kamen B A,    Colnaghi M I, Zurawski V R Jr, “Cloning of a tumor-associated    antigen: MOv18 and MOv19 antibodies recognize a folate-binding    protein”, Cancer Res. 1991, 51, 6125-6132;-   Weitman S D, Lark R H, Coney L R, Fort D W, Frasca V, Zurawski V R    Jr, Kamen B A, “Distribution of the folate receptor (GP38) in normal    and malignant cell lines and tissues”, Cancer Res., 1992, 52,    3396-3401;-   Garin-Chesa P, Campbell I, Saigo P, Lewis J, Old L, Rettig W,    “Trophoblast and ovarian cancer antigen LK26. Sensitivity and    specificity in immunopathology and molecular identification as a    folate-binding protein”, Am. J. Pathol., 1993, 142, 557-567;-   Holm J, Hansen S I, Hoier-Madsen M, Sondergaard K, Bzorek M, “Folate    receptor of human mammary adenocarcinoma”, APMIS, 1994, 102,    413-419;-   Franklin W A, Waintrub M., Edwards D, Christensen K, Prendergrast P,    Woods J., Bunn P A, Kolhouse J F, “New anti-lung cancer antibody    cluster 12 reacts with human folate receptors present on    adenocarcinoma”, Int. J. Cancer, 1994, 8 (Suppl.) 89-95.-   Miotti S, Canevari S, Menard S, Mezzanzanica D, Porro G, Pupa S M,    Regazzoni M, Tagliabue E, and Colnaghi M I, “Characterization of    human ovarian carcinoma-associated antigens defined by novel    monoclonal antibodies with tumor-restricted specificity”, Int. J.    Cancer, 1987, 39, 297-303; and-   Vegglan R, Fasolato S, Menard S, Minucci D, Pizzetti P, Regazzoni M,    Tagliabue E, Colnaghi M I, “Immunohistochemical reactivity of a    monoclonal antibody prepared against human ovarian carcinoma on    normal and pathological female genital tissues”, Tumori, 1989, 75,    510-513.

Folate binding proteins are also present in normal adult oviductepithelium and in kidney distal and proximal tubules, where they serveto prevent excessive loss of folate via the urine. Kidneys may, as aresult, be a significant source of toxicity. Folic acid in high doseshas been reported to be nephrotoxic and a kidney-specific tumorpromoter, as it is rapidly concentrated in the kidney and precipitatedin the tubules as urinary pH drops, causing obstructive nephropathy.This injury results in diffuse renal cell proliferation and hypertrophy.Rats given i.v. injections of folic acid (250 mg/kg) in 0.3 M sodiumbicarbonate showed an increase in the ratio of kidney to body weightthat reached 165% of control by 24 h after treatment. See for example:

-   Klinger E L J, Evan A P, Anderson R E, “Folic acid-induced renal    injury and repair”, Arch. Pathol. Lab. Med. 1980, 104, 87-93;-   Hsueh W. Rostorfer H H, “Chemically induced renal hypertrophy in the    rat”, Lab. Invest. 1973, 29, 547-555; and-   Dong L. Stevens J L, Fabbro D, Jaken S, “Regulation of Protein    Kinase C isozymes in kidney regeneration”, Cancer Res. 1993, 53,    4542-4549.

Overexpression of FBP by a number of different tumors has led a numberof investigators to explore its potential as a delivery system fortoxins or poorly permeable compounds coupled to folic acid and as ameans to increase selective delivery of antifolate drugs such asmethotrexate to tumors. The amount of FBP on the membrane of ovariancancer cells is high (1×10⁶ molecules/cell). IGROV cells in culture canbind ³H folic acid at a level of 10-12 pmol/10⁶ cells; MA104 cells bind1-2 pmol folic acid/10⁶ cells. FBP has a very high affinity for folicacid and some of its reduced folate cofactors (K_(d)˜1-10 nM); thispresumably favors folate uptake at the usual folate concentrations thatexist in vivo (5-50 nM). The recycling rate for the folate bindingprotein (in vitro) has been reported to range from ˜30 min in MA104cells to 5 hr in L1210 cells. Several antifolate drugs have been shownto bind to FBP; these compounds, of which methotrexate ischaracteristic, have been used to antagonize the growth of cancer cells.See, for example:

-   Orr R B, Kamen B A, “UMSCC38 cells amplified at 11q13 for the folate    receptor synthesize a mutant nonfunctional folate receptor”, Cancer    Res. 1994, 54, 3905-3911;-   Anthony A C, “The biological chemistry of folate receptors”, Blood,    1992, 79, 2807-2820; and-   Spinella M J, Brigle K E, Sierra E E, Goldman, I D, “Distinguishing    between folate receptor-α-mediated transport and reduced folate    carrier-mediated transport in L1210 leukemia cells”, J. Biol. Chem.,    1995, 270, 7842-7849.

These studies indicate an essential fact necessary to distinguishbetween normal cells and tumor cells when delivering pharmaceutical ordiagnostic agents into a patient using folates to be internalized byFBP. FBP levels are low in many normal tissue types while, incomparison, FBP levels are high in many tumor cells. This differencebetween the folate receptor levels allows selective concentration ofpharmaceutical or diagnostic agents in tumor cells relative to normalcells, thereby facilitating treatment or visualization of tumor cells.

In culture, cells were successfully targeted through FBP usingfolate-conjugated protein toxins that would not normally penetrate thecell membrane through diffusion, as well as with folate-derivatizeddrug/antisense oligonucleotide-carrying liposomes. See, for example:

-   Leamon C P, Low P S, “Cytotoxicity of momordin-folate conjugates in    cultured human cells”, J. Biol. Chem., 1992, 267, 24966-24967;-   Leamon C P, Paston I, Low P S, “Cytotoxicity of folate-pseudomonas    exotoxin conjugates towards tumor cells”, J. Biol. Chem., 1993, 268,    3198-3204;-   Lee R J, Low P S, “Delivery of liposomes into cultured KB cells via    folate receptor-mediated endocytosis”, J. Biol. Chem., 1994, 269,    3198-3204;-   Wang S, Lee R J, Cauchon G, Gorenstein D G, Low P S, “Delivery of    antisense oligonucleotides against the human epidermal growth factor    receptor into cultured KB cells with liposomes conjugated to folate    via polyethyleneglycol”, Proc. Natl. Acad. Sci U.S.A., 1995, 92,    3318-3322; and-   Wang S, Lee R J, Mathias C J, Green M A, Low P S, “Synthesis,    purification and tumor cell uptake of Ga-67-Deferoxamine-folate, a    potential radiopharmaceutical for tumor imaging”, Bioconj. Chem.,    1996, 7, 56-63.

The prior art has spent considerable energy in studying folate bindingprotein as a potential target for delivery of exogenous molecules intocells that express folate binding protein, as further illustratedhereunder.

U.S. Pat. No. 5,416,016 and WO 96/36367 (Low et al.) are directed to amethod for enhancing transmembrane transport of exogenous molecules anddisclose such delivery wherein the method comprises: contacting amembrane of a living cell with a complex formed between an exogenousmolecule and a ligand of folic acid and folate analogs to initiatereceptor-mediated transmembrane transport of the ligand complex. Theexogenous molecules include a large variety of compounds, peptides,proteins and nucleic acids, analgesics, antihypertensive agents,antiviral agents, antihistamines, cancer drugs, expectorants, vitamins,plasmids and diagnostic agents.

The synthetic methods described in these documents were notregioselective, and mixtures containing folic acid coupled to theexogenous molecule through either the α- or γ-carboxylate of folate areexpected to form. In the process disclosed in U.S. Pat. No. 5,416,016these mixtures were not separated.

WO 96/36367 distinguishes between the two isomers of DF-folates, i.e.,those where deferoxamine is coupled to the folate moiety through the α-or through the γ-carboxyl group of folate, based on their competitionwith free folate for the cell surface FBP: it was found that theax-conjugate was unable to compete with free folate for the cell surfaceFBP. In a comparative test a 50% decrease in bound [³H] folic acid wasobserved in the presence of an equimolar amount of the DF-folate (γ)conjugate, while the DF-folate (α) isomer displayed no ability tocompete with the radiolabeled vitamin.

Wang et al., supra, studied the uptake of ⁶⁷Ga-deferoxamine-folate intoKB tumor cells (a human nasopharyngeal epidermal carcinoma cell linethat greatly overexpresses the folate binding protein) as a potentialradiopharmaceutical. When 0.15 μCi (100 pmol) of ⁶⁷Ga-DF-folate(deferoxamine coupled to folic acid via the γ-carboxylate of folate) wasincubated with monolayers of KB cells, the final % uptake of thecompound by the KB cells was 32% of the applied radioactivity. Thecompound had very low non-specific binding as indicated by very lowactivity levels bound to a receptor-negative cell line control.

Wang et al. subsequently published another report* stating that folicacid derivatives that are modified at the alpha carboxylate have noaffinity for cell surface folate receptors. They reported thepreparation of FITC-EDA-folate derivatives containing a fluoresceinmoiety (FITC) linked to folate through either the α- or γ-carboxylate offolate (via an ethylenediamine [EDA] spacer). The two isomers wereincubated with KB cells that overexpress FBP. The cells were then washedto remove unbound compound and assayed for cell-associated fluorescence.The γ-isomer of FITC-EDA-folate showed half maximal binding to KB cellsat a concentration of 1.6 nM (binding comparable to native folate), butthe α-isomer of FITC-EDA-folate had “virtually no affinity for the cellsurface receptors”.

-   *Wang, Susan; Luo, Jin; Lantrip, Douglas A.; Waters, David J.;    Mathias, Carla J.; Green, Mark A.; Fuchs, Philip L.; Low, Philip S.    Design and Synthesis of [¹¹¹In]DTPA-Folate for Use as a    Tumor-Targeted Radiopharmaceutical. Bioconjugate Chem. (1997), 8(5),    673-679.

The folate-based agents of the present application were designed for usein nuclear medicine, neutron capture therapy, or MRI applications. Basedon the teachings in WO96/36367 that only folate adducts coupled toexogenous molecules through the gamma carboxylate of folate arerecognized by FBP, we devised regiospecific syntheses for thepreparation of these folate conjugates, rather than using thenon-regiospecific methods used by others. The conjugates prepared bythese regiospecific routes contained metal chelating ligands coupled tofolate through its gamma carboxylate. The corresponding alpha isomerswere prepared for use as negative controls. Surprisingly, when theability of the alpha and gamma isomers to bind to FBP in tumor cells invitro was compared, the alpha isomers (our “negative” controls) bound toFBP to the same extent as the gamma isomers in a variety of in vitrostudies (vide infra). This result was surprising in light of the reportsof Wang et al. Also surprising was our subsequent finding that folatecompounds derivatized with metal chelates at both the alpha and gammacarboxylate of folate (bis derivatives) were also able to bind to FBP.

We also performed studies with the alpha and gamma isomers intumor-bearing animals, where ability of the alpha isomers to localize inthe tumors was surprisingly found to be equal to or greater than thatobserved with the corresponding gamma conjugates. In addition, theclearance behavior of the two isomers was compared, both in vivo and invitro. As discussed in greater detail later, the urinary clearance ofthe alpha isomers from the body was significantly and unexpectedlyhigher than that observed with the corresponding γ-isomer or with ³Hfolate. This may be an advantage for some nuclear medicine andradiotherapy applications for these compounds, because retention innon-target organs causes higher radiation dose to the patient and lowertarget to background ratios. Compounds that are more rapidly excretedfrom the body provide an improved margin of safety.

We have also discovered that the alpha isomers of the folate conjugatesof the present invention also show unexpectedly faster clearance fromcells in vitro. Studies were performed to compare the clearance of metalcomplexes coupled to the γ- or α-carboxylate of folates or to both theα- and γ-carboxylates of folates (hereinafter sometimes termed bisderivatives) from KB and JAR cells. We obtained the surprising findingthat the clearance rate of the α isomer and of the bis isomer from KBand JAR cells is significantly faster than that of the corresponding γisomer or of ³H folate.

Based on this surprising discovery we have also found that the clearancerate of folate-based diagnostic agents designed for use in nuclearmedicine or MRI applications can be varied or tailor-made by usingvarious proportions of the α-isomer, the bis isomer and γ-isomer of suchdiagnostic agents. In addition to tailor-making the rate of clearancefrom certain organs, such as the kidney, liver, brain, liver, kidneysand from various tissues such as tumors that over-express folate bindingprotein, the use of chelating agents chosen for the compounds of thepresent invention provides a greater margin of safety against thetoxicity of the metal used in the chelates.

Experiments from our laboratories on the cellular uptake of monomericfolate conjugates of Gd chelates designed for use in MR applicationsindicate that structural modifications that bring about an increase theintensity of the MR signal are advantageous, as the signal intensityobtainable with this technique is determined by the quantity ofparamagnetic or superparamagnetic metal that can be localized in thetarget tissues. This is, in turn, limited by the quantity of folatebinding protein present in those tissues. The desired increase in signalintensity could be achieved by attaching multimeric Gd chelates to asingle folate residue and/or by the use of enhanced relaxivity Gdchelates, that are, as a result of their structure, expected to providehigher intrinsic signal intensity per Gd atom. Based on theseobservations the following concepts are presented for the design of newmonomeric and multimeric folate conjugates of Gd chelates in order toenable MR imaging of tumors and other tissues that over-express thefolate binding protein.

SUMMARY OF THE INVENTION

In accordance with the present invention, diagnostic and therapeuticcompositions, methods for use, and processes for their preparations areprovided. More particularly, the invention is directed to the followingmedical/pharmacological diagnostic and therapeutic areas of the art.

a) MR Diagnostic Composition for Visualization of Tissues thatOver-express FBP using MRI

The composition comprises macrocyclic and non-macrocyclic ligandschelated to superparamagnetic or paramagnetic metals and selectivelycoupled to folate-receptor binding ligands through the alpha, or boththe alpha and gamma carboxylate of the folate-receptor binding ligand.Polyaza macrocyclic ligands with enhanced relaxivity properties andcompounds that contain more than one Gadolinium per folate areespecially preferred. Derivatives of folic acid and of methotrexate(MTX) are included in the composition and use of the present invention.

Polyaza macrocyclic ligands chelated to superparamagnetic orparamagnetic metals and coupled to folate-receptor binding ligandsthrough the gamma carboxylate of folate are also included in thecomposition and use of the present invention. Enhanced relaxivityligands and ligands that contain more than one Gd per folate areespecially preferred. Derivatives of folic acid and of methotrexate(MTX) are included in the composition and use of the present invention.

b) Radiodiagnostic Composition for Visualization of Tissues usingNuclear Medicine Techniques

The composition comprises:

-   -   macrocyclic and non- macrocyclic ligands chelated to radioactive        gamma-emitting metals and coupled to folate-receptor binding        ligands through either the alpha, or both the alpha and gamma        carboxylate of the folate-receptor binding ligand; and    -   selected macrocyclic and non-macrocyclic ligands chelated to        radioactive gamma-emitting metals and coupled to folate-receptor        binding ligands through the gamma carboxylate of the        folate-receptor binding ligand. Both derivatives of folic acid        and methotrexate (MTX) are included for use in the composition.        c) Composition for Radiotherapy

The composition comprises:

-   -   macrocyclic and non-macrocyclic ligands chelated to radioactive        alpha or beta-emitting metals that are coupled to folic acid        receptor binding ligands through the alpha carboxylate or        through both the alpha and gamma carboxylate of the        folate-receptor binding ligand, and    -   selected ligands chelated to radioactive alpha or beta-emitting        metals and coupled to folate-receptor binding ligands through        the gamma carboxylate of the folate-receptor binding ligand.        Both derivatives of folic acid and of methotrexate (MTX) are        included for use in the composition.

In particular embodiments of compositions (b) and (c) the invention isdirected to a radio-diagnostic or radiotherapeutic agent comprising achelated radioactive metal complexed with a folate receptor-bindingligand, which on administration to a patient is capable of enhancing thetransport of the radioactive metal across the membrane of living cells,and of beneficially affecting the biodistribution thereof, therebyfacilitating visualization or radiotherapy of the part of the body beingexamined by nuclear medicine diagnostic or radiotherapy techniques. Asuitable radiotherapeutic composition according to the inventioncomprises, as the active ingredient, a folate-metal chelate derivativethat bears an alpha- or beta-emitter that is suitable for radiotherapy.Suitable radionuclides for radiotherapy are e.g. those that are listedin “Radionuclides for Therapy”, ed. P. Schubiger and P. H. Hasler, 1986.

In particular embodiments of composition a) the invention is directed toa paramagnetic diagnostic agent comprising a chelated paramagnetic metalconjugated to a folate receptor binding ligand, which on administrationto a patient is capable of enhancing the transport of the paramagneticmetal across the membrane of living cells, and beneficially affectingthe biodistribution thereof, thereby facilitating visualization of thepart of the body being examined by Magnetic Resonance Imaging diagnostictechniques. A second embodiment of this invention comprises a method forradiotherapy by neutron capture techniques, comprising administering toa patient said composition, wherein the metal is gadolinium and, afterlocalization in the desired tissues, irradiating said tissues withneutrons to achieve emission of Auger electrons by the gadolinium to theextent that the desired tissue is damaged.

d) Composition for Chemotherapy

The composition comprises: derivatives of folic acid (but notmethotrexate) coupled to a cancer-chemotherapy drug through the alphacarboxylate of folic acid, or coupled through both the alpha and gammacarboxylate.

In a particular embodiment of the composition, the invention is directedto a chemotherapeutic agent comprising a chemotherapeutic compoundcomplexed with a folate receptor-binding ligand through its alphacarboxylate functionality, which on administration to a patient iscapable of enhancing the transport of the chemotherapeutic agent acrossthe membrane of living cells, and decreasing the uptake to non-targetorgans thereby facilitating treatment of the tumor being targeted.

In all of these inventions, unmetallated ligand may be coinjected withthe metal complexes of the ligand to affect the biodistribution of themetal complex in a useful way such as enhanced clearance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows binding of ¹⁵³Gd-DO3A-APA-(α)-folate,¹⁵³Gd-DO3A-APA-(γ)-folate or ³H folate to KB cells at 37° C.;

FIG. 1A shows binding of ¹⁵³Gd-folates or ³H folate to JAR cells at 37°C.;

FIG. 2 shows binding of ³H-folate and of the alpha or gamma isomer of¹⁵³Gd-DO3A-APA-folate to KB cells at 4° C. in the presence and absenceof excess folate;

FIG. 3 shows washout of ¹⁵³Gd(DO3A-APA)-(α- or γ-)folate or ³H-folatefrom JAR cells;

FIG. 3A shows washout of ¹⁵³Gd(DO3A-APA)-(α- or γ-)folate or ³H-folatefrom KB cells;

FIG. 4 shows binding of ³H-folate and alpha or gamma isomer of^(99m)Tc-Oxa-folate to KB cells at 4° C. in the presence and absence ofexcess folate.

FIG. 4A shows exchange (with 250 nm cold folate in the medium) offolates from KB cells; and

FIG. 4B shows exchange (with 250 nm cold folate in the medium) offolates from JAR cells.

FIGS. 5 and 5A show the synthetic scheme for the preparation of theγ-isomer of the folic acid APADO3A conjugate 6;

FIG. 6 and 6A show the synthetic scheme for the preparation of theα-isomer of the folic acid APADO3A conjugate 10;

FIGS. 7, 7A and 7B show the synthetic scheme for the preparation of thefolic acid bis(DO3A-APA) conjugate 14;

FIG. 8 shows the synthetic scheme for the preparation of a DO1MA analog17a bearing a conjugable amino function, along with carboxyl protectionthat is necessary for conjugation, starting from DO3A-tris-t-butyl ester15a, said DO1MA analog 17a could also be prepared starting fromDO3MA-tris-t-butyl ester 15b;

FIG. 9 shows the synthetic scheme for the preparation of azido-triflate16b;

FIG. 10 shows the synthetic scheme for the preparation of the DO1MAanalog 21a or DOTMA analog 21b bearing a conjugable carboxyl functionstarting from 15a or 15b, respectively;

FIGS. 11 and 11A show the synthetic scheme for the preparation of thetriflyloxy mixedester 20;

FIG. 12 shows the synthetic scheme for the preparation of the conjugableMPDO3A analogs 27a and 27b;

FIGS. 13 and 13A show the synthetic scheme for the preparation of thealkylating agent 25;

FIGS. 14 and 14A show the synthetic scheme for the preparation of theα-folate conjugate 35a of the amino group-bearing enhanced relaxivityligand 17b;

FIG. 15 shows the synthetic scheme for the preparation of the succinicmonoamide tri-carboxylic ester 42;

FIG. 16 shows the synthetic scheme for coupling amine 38 with thecarboxylic acid 42 employing carbonyldiimidazole in dimethylformamide;

FIGS. 17 and 17A show the synthetic scheme for the preparation of theprotected amino tricarboxylic ester 44;

FIG. 18 shows the synthetic scheme for the preparation of the conjugableoxa-PnAO ligand 52b starting from the amine 50;

FIG. 19 shows the synthetic scheme for the preparation of the alkylatingagent 51;

FIG. 20 shows the synthetic scheme for the preparation of the oxa-PnAOligand 56 bearing two amino groups;

FIG. 21 shows the synthetic scheme for the preparation of the α-folateconjugate 62 of the amino group-bearing oxa-PnAO ligand 52b; and

FIG. 22 shows the synthetic scheme for the preparation of gamma folateconjugate 64.

DETAILED DESCRIPTION OF THE INVENTION

1. General Description of the Folate Conjugates

Compounds of the present invention include derivatives of folic acid andmethotrexate. The structure of folic acid is shown as FIG. Ia. Monomericfolic acid derivatives of the present invention are shown as FIG. Ib.Monomeric methotrexate derivatives of the present invention are shown asFIG. Ic.

In these structures, each X may independently be —O—, —S—, —NR—, or—NH—. The group W₁ is attached via X to the alpha carboxylate of thefolic acid or methotrexate derivative; the group W₂ is attached at thecorresponding gamma carboxylate. Metal chelating ligands (K) and theiroptional chelated metals (M) and any linking groups needed to couplethese chelates to the folate-receptor binding moiety can be attached aspart of W₁ (alpha derivatives), W₂ (gamma derivatives) or both W₁ and W₂(bis derivatives). Compounds that are derivatized at W₁ (alpha and bisderivatives) are preferred.

Several structural variations are possible with this formulation. Forexample, folate-receptor binding ligands of the present applicationcomprising a single folate-receptor binding residue that is conjugatedthrough its alpha carboxylate via an optional linking group (A)p to onemetal-chelating ligand (K₁) that is optionally chelated to one metal(M₁) could be described by the general formula shown below.

-   -   wherein R₀ is a folate-receptor binding residue of formula and        the dashed line indicates the point of attachment of R.    -   K₁ is a metal chelating ligand radical that is coupled to the        remainder of the molecule via a linking group (A)p;    -   Each X is independently —O—, —S—, —NH— or —NR—;    -   K₂ is —H, -alkyl, -alkenyl, -alkynyl, -alkoxy, -aryl, -alkyl,        —CON(R2)2, -glutamate, -polyglutamate;    -   M₁ is a metal, n₁ is 0 or 1 (0=metal absent, 1=metal present);    -   b₁ is 1 [1 receptor binding residue per chelating ligand radical        K₁]    -   m₁ is 1 [one chelating ligand per linking group (A)p]        If the gamma carboxylate of this compound was then also        derivatized with a second metal chelating group (K₅) and        chelated metal (M₅), a bis derivative of the structure below is        formed:    -   wherein n₁, n₅, b₁, b₅, m₁, and m₅ are all equal to 1.        If a single metal-chelating ligand K₁ is derivatized with 2 side        arms, each of which is coupled to a different folate receptor        binding residue through its alpha carboxylate, a different sort        of bis compound is formed, wherein n₁ and m₁=1 and b₁=2, as        shown schematically below:

The alpha and alpha/gamma (bis) derivatives of formula II of the presentapplication are generally defined as given below. In this definition,derivitization at the alpha position by a chemotherapeutic drug, ratherthan a metal-chelate is also considered.

A folate-receptor binding ligand comprising one or more folate-receptorbinding residues, at least one of which is conjugated through its alphacarboxylate via an optional linking group to

-   -   (i) one or more macrocyclic or non-macrocyclic metal-chelating        ligand radicals that are optionally chelated to paramagnetic,        superparamagnetic, radioactive or non-radioactive metals capable        of either being detected outside the body by imaging means for        diagnosis or capable of providing a therapeutic or        radiotherapeutic effect; or    -   (ii) a chemotherapeutic drug        wherein said folate receptor binding ligand has the structure of        formula II:        wherein R₀ is a folate-receptor binding residue of formula:    -   each X is independently —O—, —S—, —NH—, or —NR₁—;    -   n1 is 0 or 1;    -   b1 is 1 to 3;    -   m1 is 1 to 81;    -   each K₁ is independently        -   (a) a macrocyclic or non-macrocyclic metal-chelating ligand            radical that is optionally chelated to a paramagnetic,            superparamagnetic, radioactive or non-radioactive metal M₁,            or        -   b) a chemotherapeutic drug;    -   —K₂ is —H, -alkyl, -alkenyl, -alkynyl, -alkoxy, -aryl, -alkyl,        —CON(R₂)₂, -glutamate, -polyglutamate, or —K₃;    -   —K₃ is        wherein    -   —K₅ is either        -   (a) a macrocyclic or non-macrocyclic metal-chelating ligand            that is optionally chelated to a paramagnetic,            superparamagnetic, radioactive or non-radioactive metal M₅,            or        -   (b) a chemotherapeutic drug;    -   n5 is 0 or 1;    -   b5 is 1 to 3;    -   m5 is 1 to 81;    -   —(A)p— and —(A)p*— are each independently optional linkers        comprising a straight or branched chain wherein the moieties “A”        are the same or different and selected from the group consisting        of: —CH₂—, —CHR₃—, —CR₄R₅—, —CH═CH—, —CH═CR₆—, >CR₇—CR₈<, —C═C—,        —CR₉═CR₁₀—, —C≡C—, -cycloalkylidene-, -cycloalkenyl-,        -arylidene-, -heterocyclo-, carbonyl (—CO—), —O—, —S—, —NH—,        —HC═N—, —CR₁₁═N—, —NR₁₂—, —CS—,        and    -   p and p* are independently 0 to 24, or    -   —X—[(A)]p— and —X—[(A)p]*— may each independently be the group        —Q— wherein —Q— is        —[C(R′)(R″)]_(s1)—[C(t)(R₂₁)]_(s2)—[C(R₂₂)(R₂₃)]_(s3)—X₃—Y—X₄—;        wherein        -   each s1, s2, s3, and s4 is independently 0 to 2;        -   each X₃, X₄, X₅, and X₆ is independently a single bond, —O—,            —S—, or —N(R₂₄)—;        -   Y is a single bond, —C(R₂₅)(R₂₆)—, or Y1 wherein,            -   Y1 is —C(═X₅)—X₆—W—, wherein                -   W is a single bond, -alkylidene-, -cycloalkylidene-,                    -arylidene-, -alkenylidene-, or -alkynylidene-,                    whose carbon atoms may or may not be substituted;        -   t is H, R₂₇, —C(O)OR₂₈, —P(O)(OR₂₉))OH, —P(O)(OR₃₀))OR₃₁,            —P(O)(OR₃₂)R₃₃, —P(O)(OH)R₃₄—C(O)N(R₃₅)(R₃₆), or            —C(O)NH(R₃₇);    -   each R′ and R″ is independently a single bond, H, alkyl, alkoxy,        cycloalkyl, hydroxyalkyl, aryl, or heterocyclo, each of which is        optionally substituted,    -   each R₃ through R₅, R₇, R₈, R₂₁ through R₂₃, and R₂₅ through R₂₇        is independently H, alkyl, alkoxy, halogen, hydroxy, cycloalkyl,        hydroxyalkyl, aryl, or heterocyclo, each of which is optionally        substituted;    -   each R₁, R₂, R₆, R₉ through R₁₂, R₂₄, and R₂₈ through R₃₇ is        independently H, alkyl alkenyl, cycloalkyl, aryl, a 5- or        6-membered nitrogen or oxygen containing heterocycle;        or a salt thereof.

The compounds described above are all derivatized at the alpha positionof the folate receptor binding residue, as they all contain at least onemoiety K₁ (wherein K₁ is a macrocyclic or non-macrocyclicmetal-chelating ligand that optionally contains a paramagnetic,radioactive or non-radioactive metal, or is a chemotherapeutic drug).Alternatively, for selected metal chelating group(s) and structuralmotifs, the metal chelate(s) may be placed only at the gamma position.In these cases, W₁ of formula Ib or Ic can be a group such as H, alkyl,alkenyl, alkynyl, alkoxy, aryl, alkyl-CON(R₃)₂, glutamate orpolyglutamate.

The nature of the linking side chain (A)p can be varied. For both folicacid and methotrexate derivatives of the present application, —(A)pand/or —(A)p* are optional linkers that can be any chemical moiety whichserves to physically distance, or otherwise isolate, the metal-chelatingligand or chemotherapeutic agent from the rest of the folate bindinggroup. If p=0, then K₁ or K₂ will be directly linked to X. If p=≧1, thenA, or the various A units can form a straight or branched chain, and canbe derivatized with one or with multiple metal chelating groups. It isunderstood that p can be any convenient value depending upon the designchoices for the desired complex. Preferably, p is ≦24 and mostpreferably p≦10.

The compounds of the present application are used for the preparation ofdiagnostic, therapeutic or radiotherapeutic compositions used forvisualization, therapy or radiotherapy of tissues or organs thatoverexpress folate-binding protein. Said compositions comprising:

-   -   a) a pharmaceutically acceptable carrier; and    -   b) a folate-receptor binding ligand comprised of one or more        folate-receptor binding residues each of which is conjugated        through at least one of its carboxylate moieties via an optional        linking group to either a) one or more polydentate macrocyclic        or non-macrocyclic metal-chelating ligand residues that are        optionally chelated to radioactive or non-radioactive metals        capable of either being detected outside the body by imaging        means for diagnosis or capable of providing a therapeutic or        radiotherapeutic effect; or b) a chemotherapeutic drug.

The metal chelating groups can be either macrocyclic or non-macrocyclicmultidentate metal chelating ligands, and the structure of these ligandsand the metals that are chelated to them may be varied depending on theuse envisioned for them. For compounds of the present application thatare used for Magnetic Resonance Imaging applications, chelating polyazamacrocyclic ligands that form stable compounds with superparamagnetic orparamagnetic metals, and chelating ligands that provide enhancedrelaxivity properties (vide infra) are preferred. For such applications,gadolinium is the preferred metal.

Novel dendrimeric structures that contain multiple metal chelatinggroups can be envisioned that are especially useful for therapeutic orradiotherapeutic applications where it is useful to deliver a largequantity of the chelated metal or therapeutic drug that is used forvisualization or therapy or radiotherapy into the targeted tissue. Forexample, compounds which have the general structure depicted by formulaVII,

-   -   wherein R₀ is a folate-receptor binding residue of formula:        dendrimeric structures could be prepared such that W₁ or W₂        contains multiple metal chelating groups. For example, if either        W₁ or W₂ or both contained a radical of formula VIIIc:        wherein K₁ is a metal chelating ligand radical and M₁ is a metal        ion, the resulting dendrimeric complex could be used to deliver        high concentrations of gadolinium metal to cells that express        high levels of folate binding protein, for use in magnetic        resonance imaging applications or for subsequent neutron capture        therapy. Similarly, if derivatized with metal chelates that bind        radiotherapeutic metal isotopes, such compounds could be used to        deliver high concentrations of alpha- or beta-emitting        radionuclides for radiotherapy applications.

Several specific variations of these structures and others are describedfurther below, wherein the nature of the metal-chelating groups, metalsand linking groups are selected to fine-tune the properties of thecompound to its intended use.

2. Detailed Description of the Macrocyclic Polyaza Ligands and theirFolate Conjugation

The polyaza macrocyclic compounds described below can be used for thevisualization, therapy or radiotherapy of tissues or organs thatoverexpress folate-binding protein, depending upon what metal is used.If the compounds are derivatized with a paramagnetic metal such asgadolinium (Gd), they may be used as contrast agents for MRI techniques,after selective uptake of these compounds in tissues that overexpressfolate binding protein.

Experiments from our laboratories on the cellular uptake of monomericfolate conjugates of Gd chelates using KB cells (a cell line thatoverexpresses FBP) indicated that obtaining adequate signal intensity inMagnetic Resonance Imaging experiments with these targeted imagingagents was very challenging, and that it was an advantage to makemodifications that caused a significant increase in signal intensity.This desired increase in signal intensity could be achieved by a)attaching multiple Gd chelates to a single folate residue and/or by b)the use of enhanced relaxivity Gd chelates, which are expected toprovide higher intrinsic signal intensity per Gd atom. The use of morethan one folate residue per molecule also appears to be propitious basedon the work of E. C. Wiener et al., Investigative Radiology, 1997, 32,748-7544, who estimated that greater than ˜10 gadolinium atoms perfolate would be required for successful contrast enhancement in magneticresonance imaging.

Based on these observations, the following concepts are presented forthe design of new monomeric and multimeric folate conjugates of Gdchelates in order to enable MR imaging of tumors that over-express thefolate binding protein. Said chelates can also be used inradiodiagnostic and radiotherapeutic techniques, if a suitableradioactive metal is substituted for gadolinium.

A) General Structures for Monomeric and Multimeric Polyaza-macrocyclicLigands Conjugated to Folate Moieties

The structures disclosed are further modifications of ligand motifs thathave been demonstrated to possess enhanced relaxivity as discussed inour co-pending applications WO 95/31444 (Nov. 23, 1995), Ser. No.08/010,909 (Jan. 29, 1993), U.S. Pat. Nos. 5,573,752, and 5,358,704. Theaim of making the modifications is to enable conjugation of suchenhanced relaxivity ligands to targeting vectors such as folate receptorbinding compounds. The relaxivity of a paramagnetic compound is ameasure of its signal enhancing effect when used as a contrast agent forMRI. Enhanced relaxivity compounds provide a stronger signal enhancingeffect per molecule than can be obtained with the typical relaxationagents that are used for contrast enhancement. We have found thatcertain macrocyclic metal-chelating ligand motifs, when chelated toparamagnetic metals such as gadolinium, provide an unexpectedly strongsignal-enhancing effect. If such enhanced relaxivity chelates areincorporated into a compound that targets a particular tissue such asthe folate receptor, localization at the target results in a highersignal intensity than can be obtained if the comparable compound werederivatized with normal chelates for Gd, such as DTPA.

The amine-thiocarboxylate and carboxylate-containing macrocyclesdepicted by formula VIa below are conjugatable enhanced relaxivitymotifs that can be used for coupling to targeting vectors such asfolate. These intermediates are an integral part of this invention, andcan be used to prepare conjugates that contain one, or preferablygreater than one metal chelate per folate residue. Such multimericcompounds are particularly useful, as multiple paramagnetic metals arelocalized at the target tissue upon binding of a single folate receptorbinding moiety. The presence of multiple paramagnetic metal chelates permolecule, coupled with the enhanced relaxivity properties provided byeach of these metal chelates, should significantly improve thesensitivity of magnetic resonance imaging agents designed for thedetection of folate-receptor positive tissue.

(i) Intermediate Ligands Bearing Free Carboxylate, Thiocarboxylate orAmino Functions for Conjugation to Folate Moieties

The conjugatable polyaza macrocyclic ligands are depicted by formulaVIa. These intermediates contain at least one free amine, carboxylate orthiocarboxylate functionality that can be used for conjugation totargeting vectors such as folate.

wherein

-   -   n is 0 or 1;    -   each m, o, and p is independently 1 or 2;    -   —Q(int) is a conjugatable amine-, carboxylate- or        thiocarboxylate-containing group of formula        —[C(R′)(R″)]s₁—[C(t)(R₂₁)]s₂—[C(R₂₂)(R₂₃)]s₃—X₃—Y—X₄; wherein        -   s1, s2, s3, and s4 are independently 0 to 2;        -   X₃ is a single bond, —O—, —S—, —NH— or —NR₂₄— if Y is            present, or X₃ is —OH, —SH, —NH₂ or —N(R₂₄)H if Y and X₄ are            absent;        -   X₄ is a single bond, —OH, —COOH, —SH, —NHR₂₄ or —NH₂;        -   Y is a single bond, —C(R₂₅)(R₂₆)—, or Y1            wherein,    -   Y1 is —C(═X₅)—X6—W—, wherein        -   X₅ is ═O or ═S;    -   X₆ is a single bond, —SH, —NH(R₃₈), —NH₂ or —OH if W and X₄ are        absent, and is —S—, —O—, —NH—, or —N(R₃₉)—, if W and X₄ are        present;        -   W is a single bond, or is -alkylidene-, -cycloalkylidene-,            -arylidene-, -alkenylidene-, or -alkynylidene-, whose carbon            atoms may or may not be substituted;        -   t is —H, —R₂₇, —C(O)OR₂₈, —P(O)(OR₂₉))OH, —P(O)(OR₃₀))OR₃₁,            —P(O)(OR₃₂)R₃₃, —P(O)(OH)R₃₄—C(O)N(R₃₅)(R₃₆), or            —C(O)NH(R₃₇);        -   each —G is independently —C(O)OR′″, —P(O)(OR′″)OH,            —P(O)(OR′″)₂, —P(O)(OR′″)R″, —P(O)(OH)R″—C(O)N(R′″)₂, or            —C(O)NH(R′″);            -   each —R′ and —R″ is independently a single bond, —H,                -alkyl, -alkoxy, -cycloalkyl, -hydroxyalkyl, -aryl, or                -heterocyclo, each of which is optionally substituted,            -   each —R′″ is independently —H, -alkyl, -cycloalkyl,                -hydroxyalkyl, -aryl, or -heterocyclo, each of which is                optionally substituted,            -   each —R₁₃ through —R₂₃, and —R₂₅ through —R₂₇ is                independently —H, -alkyl, alkoxy, -halogen, -hydroxy,                -cycloalkyl, -hydroxyalkyl, -aryl, or -heterocyclo, each                of which is optionally substituted;            -   each —R₂₄, and —R₂₈ through —R₃₉ is independently —H,                -alkyl, -alkenyl, cycloalkyl, aryl, a 5- or 6-membered                nitrogen or oxygen containing heterocycle, each of which                is optionally substituted;            -   or R₁₃ together with R₁₅, and R₁₇ together with R₁₈,                independently form, together with the carbon atoms in                the polyazamacrocycle to which they are attached, a                fused fully or partially saturated non-aromatic                cyclohexyl ring which may be unsubstituted or                substituted by one or more halogen, alkyl, ether,                hydroxy, or hydroxyalkyl groups, and which may be                further fused to a carbocyclic ring, or R₁₃ and R₁₅ are                each hydrogen and R₁₇, together with R₁₈, forms a fused                fully or partially saturated non-aromatic cyclohexyl                ring as defined above, or R₁₃, together with R₁₅, forms                a fused fully or partially saturated non-aromatic                cyclohexyl ring as defined above, and R₁₇ and R₁₈ are                hydrogen;                or a salt thereof.                (ii) Monomeric Conjugates Bearing one Folate and one or                two Polyaza Macrocyclic Ligand Moieties

The conjugatable intermediates of formula VIa can be used to prepare themonomeric folate receptor binding conjugates of formula II:

wherein R₀ is a folate-receptor binding residue of formula:

-   -   each X is independently —O—, —S—, —NH—, or —NR₁—;    -   n1 is 0 or 1;    -   b1 is 1-3;    -   m1 is 1    -   K₁ is —H, -alkyl, -alkenyl, -alkynyl, -alkoxy, -aryl, -alkyl,        —CON(R₂)₂, -glutamate,- or polyglutamate, or a metal chelating        ligand radical of formula VI:    -    that is optionally chelated to a radioactive or paramagnetic        metal;    -   K₂ is —H, -alkyl, -alkenyl, -alkynyl, -alkoxy, -aryl, -alkyl,        —CON(R₂)₂, -glutamate, -polyglutamate, or    -   wherein        -   K₅ is a macrocyclic ligand radical of formula VI that is            optionally chelated to a radioactive or paramagnetic metal            M₅; with the proviso that at least one K₁ or K₂ must contain            a ligand of formula VI;        -   n5 is 0 or 1;        -   b5 is 1;        -   m5 is 1;        -   —(A)p— and —(A)p*— are —Q—;        -   Q is            —[C(R′)(R″)]_(s1)—[C(t)(R₂₁)]_(s2)——[C(R₂₂)(R₂₃)]_(s3)—X₃—Y—X₄—;            wherein            -   s1, s2, s3, and s4 are independently 0 to 2;            -   X₃, X₄, X₅, and X₆ are independently a single bond, —O—,                —S—, —NH, or —N(R₂₄)—;            -   Y is a single bond, —C(R₂₅)(R₂₆)—, or Y₁ wherein,            -   Y₁ is —C(═X₅)—X₆—W—, wherein                -   W is a single bond, -alkylidene-, -cycloalkylidene-,                    arylidene-, -alkenylidene-, or -alkynylidene-, whose                    carbon atoms may or may not be substituted;                -   t is H, R₂₇, —C(O)OR₂₈, —P(O)(OR₂₉))OH,                    —P(O)(OR₃₀))OR₃₁, —P(O)(OR₃₂)R₃₃,                    —P(O)(OH)R₃₄—C(O)N(R₃₅)(R₃₆), or C(O)NH(R₃₇);    -   each G is independently —C(O)OR′″, —P(O)(OR′″)OH, —P(O)(OR′″)₂,        —P(O)(OR′″)R′″, —P(O)(OH)R′″—C(O)N(R′″)₂, or —C(O)NH(R′″);    -   each —R′ and —R″ is independently a single bond, —H, -alkyl,        -alkoxy, -cycloalkyl, -hydroxyalkyl, -aryl, or -heterocyclo,        each of which is optionally substituted,    -   each —R′″ is independently —H, -alkyl, -cycloalkyl,        -hydroxyalkyl, -aryl, or -heterocyclo, each of which is        optionally substituted,    -   each —R₁₃ through —R₂₃, and —R₂₅ through —R₂₇ is independently        —H, -alkyl, alkoxy, -halogen, -hydroxy, -cycloalkyl,        -hydroxyalkyl, -aryl, or -heterocyclo, each of which is        optionally substituted;    -   each —R₂₄, and —R₂₈ through —R₃₉ is independently —H, -alkyl,        -alkenyl, cycloalkyl, aryl, a 5- or 6-membered nitrogen or        oxygen containing heterocycle, each of which is optionally        substituted;        -   or R₁₃ together with R₁₅, and R₁₇ together with R₁₈,            independently form, together with the carbon atoms in the            polyazamacrocycle to which they are attached, a fused fully            or partially saturated non-aromatic cyclohexyl ring which            may be unsubstituted or substituted by one or more halogen,            alkyl, ether, hydroxy, or hydroxyalkyl groups, and which may            be further fused to a carbocyclic ring, or R₁₃ and R₁₅ are            each hydrogen and R₁₇, together with R₁₈, forms a fused            fully or partially saturated non-aromatic cyclohexyl ring as            defined above, or R₁₃, together with R₁₅, forms a fused            fully or partially saturated non-aromatic cyclohexyl ring as            defined above, and R₁₇ and R₁₈ are hydrogen;            or a salt thereof.

Compounds of formula II that contain metal-chelating ligand radicals offormula VI having enhanced relaxivity properties are especiallypreferred.

(iii) Multimeric (dendrimeric) Conjugates Bearing one Folate and morethan one Polyaza Macrocycle Moiety

The dendrimeric conjugates described herein contain multiple metalchelating ligands. Such multimeric compounds are particularly useful ascontrast agents for MRI, as multiple paramagnetic metals can belocalized at the target tissue upon binding of a single folate receptorbinding moiety. If these metal chelates are chosen to have enhancedrelaxivity properties, said dendrimers may provide improved sensitivityif used as MRI contrast agents designed for the detection offolate-receptor positive tissue, or improved efficacy if used forneutron capture therapy, due to the increased concentration ofgadolinium in the cells. However, it is understood that said structurescould also be prepared using ligands that are suitable for chelation toradioactive metals, for use in radiotherapy or radiodiagnosis.

Said dendrimeric conjugates are represented by formulae VIIa-VIId, allof which have the general structure depicted by formula VII:

-   -   wherein R₀ is a folate-receptor binding residue of formula:        Variations in W₁ and W₂ (denoted by formulas VIIa-VIId below)        represent dendrimers of generations 1, 2, 3, and 4,        respectively. Such dendrimeric structures allow the        incorporation of multiple metal chelating residues per molecule.        Compounds where the metal-chelating ligand radical is present on        W₁ and not on W₂ (alpha derivatives) are preferred. In the        description below, the metal chelating radical is a derivative        of the macrocyclic ligand intermediates of formula VIa. However,        other ligand systems are also envisioned.        a. Dendrimeric Conjugates of Formula VIIa-VIId Bearing one        Folate Residue and More than one Metal Chelating Residues

(1) Ratio 1:3 or 1:6 dendrimer VIIa of the first generation:

These are described by formula VII:

wherein

-   -   for the first generation dendrimers of formula VIIa, bearing one        folate-receptor binding residue and 3 or 6 metal chelating        ligand radicals:        -   W₁ and W₂ of formula VII are each independently —OR′″,            —SR′″, —NR′″R′″—CON(R₂)₂, -glutamate, -polyglutamate, or            —K₆; wherein each —R′″ is independently —H, -alkyl, -aryl,            -cycloalkyl, -hydroxyalkyl, or -heterocyclo;        -   with the proviso that either W₁, W₂, or both W₁ and W₂ of            formula VIIa must be —K₆, where —K₆ is a residue of formula            VIIIa:            wherein    -   Y is a single bond or —Y′—C(═X)—wherein        -   X is ═O or ═S;        -   Y′ is N(R₆)—Z—; wherein            -   Z is a single bond, -alkylidene-, -vinylidene-,                -cycloalkylidene-, or -arylidene-;            -   A is —C(═O)—, C(═S), or —CH₂—N(R₇)—;            -   M₁ is a superparamagnetic, paramagnetic, radioactive or                non-radioactive metal that is optionally bound to K₁;            -   K₁ is a macrocyclic metal chelating ligand radical of                formula VI;            -   that is attached through the free —N(R)— atom of the                function Q if A is —C(O)— or —C(S)—, or through the free                —C(O)— atom of the function Q if A is —CH₂—N(R₇)—;            -   R₁ to R₇ is H, alkyl, hydroxyalkyl, alkoxy, alkoxyalkyl,                cycloalkyl, or aryl.

(2) Ratio 1:9 or 1:18 dendriner VIIb of the second generation

These are described by formula VIIb:

wherein

-   -   W₁ and W₂ of formula VII are each independently —OR′″, —SR′″,        —NR′″R″″, or —K₇, and —K₇ is a residue of formula VIIIb;    -   with the proviso that either W₁, W₂, or both W₁ and W₂ must be        —K₇ (a residue of formula VIIIb):        wherein    -   X, Y, X′, Z, A, K₁, M₁, R′″ and all R groups are defined as in        formula VIIa;        -   D is —N(R₆)—C— if A is —C(O)— or —C(S)— and            —C(═X₂)—E—N(R₇)—C— if A is —CH₂—N(R₇)—;            -   wherein E is a single bond, alkylidene, vinylidene,                cycloalkylidene, or arylidene and X₂ is ═O or ═S;

(3) Ratio 1:27 or 1:54 dendrimer VIIc of the third generation

These are described by formula VIIc

wherein

-   -   W₁ and W₂ of formula VII are each independently —OR′″, —SR′″,        —NR′″R′″, or —K₈; wherein —K₈ is a residue of formula VIIIc;        -   with the proviso that either W₁, W₂, or both W₁ and W₂ of            the compounds of formula VIIc must be —K₈:            wherein,    -   X, Y, X′, Z, A, K₁, M₁, R′″ and all R groups are defined as in        formula VIIa;        -   D₁ and D₂ are independently —N(R₆)—C— if A is C(O) or C(S),            and —C(═X₂)—E—N(R₇)—C if A is —CH₂—N(R₇)—;            -   wherein E is a single bond, alkylidene, vinylidene,                cycloalkylidene, or arylidene and X₂ is ═O or ═S;

(4) Ratio 1:81 or 1:162 dendrimer VIId of the fourth generation

These are described by formula VIId

wherein

-   -   W₁ and W₂ of formula VII are each independently —OR′″, —SR′″,        —NR′″R′″ or —K₉;        -   wherein —K₉ is a residue of formula VIIId;        -   with the proviso that either W₁, W₂, or both W₁ and W₂ must            be —K₉ (a residue of formula VIIId):        -    wherein,            -   X, Y, X′, Z, A, K₁, M₁, R′″ and all R groups are defined                as in formula VIIa;            -   D₁, D₂, and D₃ are independently —N(R₆)—C— if A is                —C(O)— or —C(S)—, and —C(═X₂)—E—N(R₇)—C if A is                —CH₂—N(R₇)—;                -   wherein E is a single bond, alkylidene, vinylidene,                    cycloalkylidene, or arylidene and X₂ is ═O or ═S;                    b. Multimeric Conjugates Bearing more than one                    Folate and Polyaza Macrocyclic Ligand Residues

Dendrimeric conjugates of this type are depicted by formulae IXa, IXb,IXc, and IXd representing dendrimers of generations 1, 2, 3, and 4,respectively.

(1) Ratio 3:3 dendrimer IXa of the first generation

Dendrimers with a ratio of three folate receptor binding residues tothree metal chelating residues are depicted by formula IXa:

wherein

-   -   F is a folate-receptor binding residue of formula:        wherein    -   R₀ is a residue of formula:    -   dentoes the point of attachment of the residues above.    -   X₁ and X₂ are independently ═O or ═S;    -   A is —C(O)—, —C(S)— or —CH₂—N(R₇)—;    -   B is a metal chelating ligand radical of formula VI attached        through the free N atom of the function —Q— if A is —C(O)— or        through the free C(O) atom of the function Q if A is        —CH₂—N(R₇)—;        -   E is a single bond, alkylidene, vinylidene, cycloalkylidene,            or arylidene;        -   —R₁, —R₆ through —R₈, —R₁₃, and —R₁₄ are independently —H,            -alkyl, -hydroxyalkyl, -cycloalkyl, or -aryl;        -   —R₂ through —R₅ and —R₉ through —R₁₂ are independently —H,            -alkyl, -hydroxyalkyl, -alkoxy, -hydroxyalkyl, -halogen,            -cycloalkyl, -aryl or -heterocyclo;            (2) Ratio 9:9 Dendrimner IXb of the Second Generation

Dendrimers with a ratio of nine folate receptor binding residues to ninemetal chelating ligand residues are depicted by formula IXb:

wherein

-   -   A, B, E, F, X₁ through X₄ are as defined for the compounds of        formula IXa;    -   D₁ and D₂ are each independently —N(R₆)—C— if A is C(O) or        C(S)—, and —C(═X₃)—E—N(R₇)—C if A is —CH₂—N(R₇)—;    -   R₁ to R₁₄ is H, alkyl, hydroxyalkyl, alkoxy, alkoxyalkyl,        cycloalkyl, or aryl.        (3) Ratio 27:27 Dendrimer IXc of the Third Generation

Dendrimers with a ratio of 27 folate receptor binding residues to 27metal chelating ligand residues are depicted by formula IXc:

wherein

-   -   D₁, D₂, D₃, and D₄ are independently —N(R₆)—C— if A is C(O) or        —C(═X₃)—E—N(R₇)—C if A is —CH₂—N(R₇)—; and all other groups are        as defined above.        (4) Ratio 81:81 Dendrimer IXd of the Fourth Generation

Dendrimers with a ratio of 81 folate receptor binding residues to 81metal chelating residues are depicted by formula IXd:

wherein

-   -   A, B, E, F, K₁, M₁ and all —R groups are is defined as in        formula IXc;    -   X₁, X₂ and X₃ are independently ═O or ═S; and    -   D₁, D₂, D₃, D₄, D₅, and D₆ are independently —N(R₆)—C— if A is        C(O) or C(S), and —C(═X₃)—E—N(R₇)—C if A is —CH₂—N(R₇)—;

It is readily conceivable that those skilled in the art could visualizedendrimers of higher generations and also dendrimers having anycombinations of folate residues and polyaza macrocyclic ligand residuesby the appropriate choice of precursors. Though such structures are notspecifically shown, the scope of the present invention will encompasssuch structures. In addition, it should be obvious that these dendrimerscould be prepared with metal chelating ligands other than the polyazamacrocycles shown here.

(B) Methods for the Preparation of Folate Conjugates with PolyazaMacrocyclic Ligands

(i) Preparation of the DOTA monoamide conjugates of the PresentInvention

a. The APADO3A γ-folate conjugate 6

The synthetic scheme for the preparation of the γ-isomer of the folicacid APADO3A conjugate 6 is given in FIGS. 5 and 5A. It is to beunderstood that other ligands can be complexed with the γ-carboxylate ofthe folic acid analogously to that of DO3A.

APADO3A tris-t-butyl ester 2 was coupled with the α-carboxy protectedglutamate derivative 1 to obtain 3. Deprotection and further couplingwith the pteroic acid derivative 4 provided 5. Successive deprotectionsfinally furnished the desired γ-folate conjugate 6.

b. The α-folate conjugate 10

The synthetic scheme for the preparation of the α-isomer of folic acidAPADO3A conjugate 10 is given in FIGS. 6A and 6B. It is to be understoodthat other chelating ligands can be conjugated to the α-carboxylate offolic acid analogously to that of DO3A.

APADO3A tris-t-butyl ester 2 was coupled with the γ-carboxy protectedglutamate derivative 7 to obtain 8. Deprotection and further couplingwith the pteroic acid derivative 4 provided 9. Successive deprotectionsfinally furnished the desired α-folate conjugate 10.

c. The α,γ-bis folate conjugate 14.

The synthetic scheme for the preparation of the folic acid bis(DO3A-APA)conjugate 14 is given in FIGS. 7, 7A, and 7B. It is to be understoodthat other macrocyclic or non-macrocyclic metal chelating ligands couldbe conjugated to the (α)- or (γ)- or both carboxylates of the folic acidanalogously to that of DO3A, using coupling, protection andde-protection schemes well known to those skilled in the art.

APADO3A tris-t-butyl ester 2 was coupled with the N-protected glutamatederivative 11 to obtain 12. Deprotection and further coupling with thepteroic acid derivative 4 afforded 13. Successive deprotections finallyfurnished the desired α, γ-bis folate conjugate 14.

(ii) Preparation of Conjugatable Enhanced Relaxivity Polyaza MacrocyclicLigands

Our co-pending application WO 95/31444, published Nov. 23, 1995, teachesthat the property of enhanced relaxivity is conferred on a molecule bythe substitution of methyl groups on 3 or 4 of the macrocyclic carbonatoms and/or the carboxymethyl arms of DOTA or DO3A. In this manner theenhanced molecules M4DOTA, M4DO3A, DOTMA, DO3MA, and M4DOTMA areobtained. Replacement of one of the carboxymethyl arms of DOTA by thephosphonomethyl arm also provides the enhanced relaxivity moleculeMPDO3A. The present invention will show pathways for the preparation ofconjugatable enhanced relaxivity molecules based on these structures.

A DO1MA analog 17a bearing a conjugatable amino function, along withcarboxyl protection that is necessary for conjugation, could be preparedstarting from DO3A-tris-t-butyl ester (15a) as shown in FIG. 8. A DOTMAanalog 17b bearing a conjugatable amino function, along with carboxylprotection that is necessary for conjugation, could also be preparedstarting from DO3MA-tris-t-butyl ester (15b) as shown in FIG. 8.

The preparation of 15a is described in U.S. Pat. No. 5,573,752 byRanganathan et al. DO3MA is described in S. I. Kang et al., Inorg.Chem., 1993, 32, 2912-2918. DO3MA-tris-t-Bu ester 15b could be preparedfrom DO3MA by treatment with isobutylene in the presence of catalyticamounts of concentrated H₂SO₄. Alternatively, 15b could be made from1,4,7,10-tetraazacyclododecane by tris-alkylation with t-butyl2-triflyloxy-D-lactate following the methodology of S. I. Kang et al.(loc cit). t-Butyl 2-triflyloxy-D-lactate is readily obtained fromcommercially available t-butyl (D)-lactate by triflylation with triflicanhydride.

The preparation of the conjugatable ligand 17a has been achieved by thealkylation of 15a with methyl 3-azido-2-triflyloxy-propionate (16b)followed by catalytic hydrogenation in the presence of Pd/C catalyst.Similar alkylation of 15b with 16b is expected to afford theconjugatable ligand 15b bearing the amino function.

The azido-triflate 16b was prepared as shown in FIG. 9. Isoserine (18)was esterified by treatment with MeOH in the presence of concentratedHCl to obtain 19. The diazo transfer reaction on 19 by treatment withtriflyl azide in the presence of Cu²⁺ ion as described by P. B. Alper etal. in Tetrahedron Letters 1996, 37, 6029-6032, followed by triflylationby treatment with triflic anhydride and 2,6-lutidine gave theazido-triflate 16b.

The DO1MA analog 21a or DOTMA analog 21b bearing a conjugatable carboxylfunction could be prepared starting from 15a or 15b, respectively, asshown in FIG. 10. For example, alkylation of 15b by the mixed diester,t-butyl benzyl 2-triflyloxy-malate 20, followed by debenzylation,employing catalytic hydrogenolysis, is expected to afford the enhancedrelaxivity DOTMA analog 21b bearing the carboxyl function.

The triflyloxy mixed ester 20 could be readily made as shown in FIGS. 11and 11A. t-Butyl malate 23 is made from malic acid (22) following theprocedure described by N. Balcheva et al. in Eur. Polym. J., 1991, 27,479-482. Selective benzylation of 23 by treatment with benzyl chlorideand triethylamine, following the procedure described in S. I. Kang etal., Inorg. Chem., 1993, 32, 2912-2918 in the case of lactic acid,followed by triflylation with triflic anhydride and 2,6-lutidine isexpected to furnish the synthon 20.

The carboxyl group bearing enhanced relaxivity DOTMA analog 21a or 21bcould be readily converted into an amino group bearing ligands 24a or24b, respectively, by first coupling 21a or 21b with a mono-protectedethylenediamine derivative such as ZNHCH₂CH₂NH₂ using HATU anddiisopropylethylamine and then removing the Z group by catalytichydrogenolysis in the presence of Pd/C catalyst.

Conjugatable MPDO3A analogs 27a and 27b containing the carboxyl groupare also accessible by methods shown in FIG. 12.

Alkylation of compound 15a by the triflate 25 is expected to provide theorthogonally protected ligand 26a. Debenzylation of 26a by catalytichydrogenolysis will provide the carboxyl group containing enhancedrelaxivity MPDO3A ligand 27a. Similar alkylation of 15b by the triflate25 is expected to afford the MPDO3A analog 27b via the benzyl ester 26b.

The alkylating agent 25 that is necessary for the above transformationscould be prepared as shown in FIGS. 13 and 13A. Benzyl 4-Hydroxybutyrate(28) is prepared from 4-hydroxybutyric acid by selective benzylationwith benzyl bromide as described in S. I. Kang et al., Inorg. Chem.,1993, 32, 2912-2918 in the case of lactic acid.

Oxidation of 28 by treatment with pyridinium chlorochromate will affordthe aldehyde 29. Successive treatment of 29, first withtriethylphosphite and then with triflic anhydride in the presence of ahindered base such as diisopropylethyl amine at low temperature isexpected to furnish the trifluoromethanesulfonyloxy derivative 25.

The carboxyl group bearing enhanced relaxivity MPDO3A analogs 27a and27b could be converted into amino group bearing ligands 30a and 30b,respectively, by first coupling 27a or 27b with a mono-protectedethylenediamine derivative such as ZNHCH₂CH₂NH₂ using HATU anddiisopropylethyl amine, followed by catalytic hydrogenolysis in thepresence of Pd/C catalyst.

(ii) Preparation of Monomeric Folate Conjugates with Enhanced RelaxivityPolyaza Macrocyclic Ligands

The α-folate conjugate 35a of the amino group-bearing enhancedrelaxivity ligand 17b could be prepared as shown in FIGS. 14 and 14A.Coupling ligand 17b with the γ-protected glutamate derivativeZNH-E(OtBU)-OH (7) using HATU and diisopropylethylamine in a solventsuch as dimethylformamide is expected to furnish the product 31.

Removal of the benzyloxycarbonyl group of 31 and further coupling withN-trifluoroacetyl-pteroic acid (4) employing DCC and HOBT, followed bysequential deprotection with piperidine, aqueous base, and finallyethanolic HCl, is expected to provide the desired folate conjugate 35a.

The corresponding γ folate conjugate 35b can be prepared by a similarapproach starting from the α protected glutamate derivative ZNH-E-(OtBu)(1). The α, γbis-conjugate 36 could also be made by coupling folic aciddirectly with two equivalents of the ligand 17b as per methods describedabove for the protected glutamic acid derivatives. Other enhancedrelaxivity ligands such as 24 and 30 could be substituted for 7 in theabove reactions to obtain the corresponding folate conjugates.

(iii) Preparation of Multimeric Folate Conjugates with EnhancedRelaxivity Polyaza Macrocyclic Ligands

As discussed above, preparation of multimeric folate conjugates of Gdchelates could deliver a higher Gd concentration into the target cells,thereby increasing the signal intensity during MR imaging. The synthesisof suitable linkers for the preparation of such compounds will first bepresented followed by the conjugation methods to obtain the folateconjugates.

a. Linker chemistry

Dendrimeric linkers are well known in literature. For example D. A.Tomalia and J. R. Dewald present examples of star burst dendrimers inU.S. Pat. No. 4,631,337. Smart cascade polymers are described by J. K.Young et al., Macromolecules, 1994, 27, 3464-3471. In the presentinvention the nitro-tris-carboxylate 37 and the tris-BOC protectedtetra-amine 38, described by J. K. Young et al. (loc. cit.), are used asstarting materials to develop a novel orthogonally protectedtris-amino-tris carboxylate derivative 39.

The preparation of the succinic monoamido tri-carboxylic ester 42 wascarried out as shown in FIG. 15.

Treatment of the nitro-tris-tBu ester 37 with trifluoroacetic acidfollowed by alkylation with benzyl bromide in pyridine provided thenitro-tris-benzyl ester 40. Reduction of the nitro group with Al/Hg gavethe amine 41, which upon treatment with succinic anhydride in pyridineprovided the succinic mono-amide 42.

Coupling of the amine 38 with the carboxylic acid 42 employingcarbonyldiimidazole in dimethylformamide, as shown in FIG. 16, yieldedthe orthogonally protected tris amino-tri carboxylate linker molecule39.

b. Preparation of multimeric folate conjugates

The multimeric folate conjugate with enhanced relaxivity ligands, inwhich one folate moiety will be linked to three enhanced relaxivityligand moieties, could be made as follows.

The linking molecule viz., the protected amino tricarboxylic ester 44,that is necessary for this preparation was made as shown in FIGS. 17 and17A. The nitro-tris-t-butyl ester 37 was reduced to the correspondingamino-tris-t-butyl ester by catalytic hydrogenation using Raney Nicatalyst and then protected as the Z derivative to obtain thetris-t-butyl ester 43. Trifluoroacetic acid deprotection furnished the Zprotected mono-amino-tris-carboxylic acid 44.

Coupling of the tris-acid 44 with the enhanced relaxivity ligand 15b,deprotection of the amino group by catalytic hydrogenolysis, and furthercoupling with the γ-tBu protected glutamate derivative 7 will give theprotected glutamide 45.

Removal of the Z group in compound 45, further coupling with the pteroicacid derivative 4, and deprotection employing successively piperidine,aqueous base, and finally TFA will afford the desired multimeric folateconjugate 46. The corresponding γ folate analog can be made startingfrom the α protected glutamate derivative ZNH-E-(OtBu) (1).

A multimeric folate conjugate with enhanced relaxivity ligands in whichthree folate moieties are linked to three ligand moieties could beprepared from the 3:3 linker 39, described above. Compound 39 could besubjected to catalytic hydrogenation in the presence of Pd/C to obtainthe tris-acid 47.

Coupling with three equivalents of ligand 15b, followed by selectivedeprotection of the BOC groups in the presence of t-Bu esters, asdescribed by F. S. Gibson et al., J. Org. Chem. 1994, 59, 3216-3218,will provide the tris amine 48.

Coupling of the tris-amine 48 with 7, deprotection of the amino group,coupling with three equivalents of the pteroic acid derivative (4), anddeprotection, employing successively piperidine, aqueous base, andfinally TFA, will afford the desired multimeric folate conjugate 49. Thecorresponding γ folate analog can be made starting from the -α-protectedglutamate derivative ZNH-E-(OtBu) (1).

(iv) Alternate Molecular Designs for Folate Conjugated PolyazaMacrocyclic Ligands

The methods provided for the monomeric and multimeric folate conjugatesof enhanced relaxivity ligands are by way of examples only. It isunderstood that other multimers of different ratios of folate to theligand and of higher generations could be made by the methods describedthat are well known to those skilled in the art and they all will fallwithin the scope of the present invention. These new conjugates areintended for the MRI imaging of tumors that over-express the folatebinding protein.

To increase the [Gd] at the folate site in vivo it is also possible tovisualize multimers other than those based on the dendrimeric linkerspresented above. For example the Gd chelate can be incorporated intonaturally occurring or unnatural amino acids carrying linkablefunctionalities such as carboxy or amino groups. These chelate bearingamino acids can be converted into peptides by methods well known tothose familiar with peptide synthesis. The fenestrae of the capillarieswill allow a size of up to about decameric Gd chelate polymers as linearpolymers and about the same size as cyclic or globular polymers.

Specifically Gd chelates containing the amino acid building blocks asfree amino acids attached to, for example, Gd(R-DO3A) where R contains aGd-binding oxygen atom capable of forming a five membered chelate ringwith one nitrogen of the DO3A macrocycle, and a free amino acid, can besynthesized into multimers of from 5 to 20 units, with about 10 unitsbeing preferred, using an automated amino acid synthesizer such as anAdvanced Chemtech 57. The terminal amino acid may be conjugated to thefolate in the ways presented elsewhere herein. For cyclic peptides alysine is inserted (unconjugated with Gd chelate, and the gamma aminogroup of the lysine can contain the folate targeting vector. Suchbifunctional peptides are also part of this invention.

3. Detailed Description of the Oxa-PnAO Ligands and their FolateConjugates

For imaging of tissues that overexpress folate binding protein usingnuclear medicine techniques, ligands that can chelate 99 m-technetiumare preferred, as this radioisotope has imaging characteristics that areoptimal for detection by commercially available gamma cameras.Experiments from our laboratories on the cellular uptake of folateconjugates of technetium (Tc) chelates using KB cells indicate that,surprisingly, localization of such conjugates in tumors that overexpressfolate binding protein is feasible using either the α- or γ-isomer ofoxa-PnAO folate. Radioimaging studies with these compounds infolate-deprived tumor-bearing mice showed good localization of the alphaderivative in tumor and kidneys, with negligible uptake by liver.Detailed description of these chelates and methods for their synthesisare now described.

A. General Structures for Oxa-PnAO Ligand Intermediates for ConjugationConjugated Folate Moieties

The structures disclosed are further modifications of ligand motifs thatwere described by Ramalingam et al. in U.S. Pat. No. 5,627,286. The aimof making the modifications is to enable conjugation to targetingvectors such as folates. Derivatives of these intermediates whereinfolic acid and methotrexate are coupled to these ligands through thealpha carboxylate have the general formulae IIIa, IIIb, and IIIc:

-   -   wherein Q is the group —(C(RR))_(m1)—Y¹        (C(RR))_(m2)—(Y²—(C(RR))_(m3))_(n)—, wherein        -   Y¹ and Y² are independently —CH₂—, —NR—, —O—, —S—, —SO—,            —SO₂— or —Se—;        -   n is 0 or 1; and m1, m2 and m3 are integers independently            selected from 0 to 4, provided that the sum of m1 and m2 is            greater than zero;    -   all R and R* groups are independently —R⁴, —Cl, —F, —Br, —OR⁵,        —COOR⁵, —CON(R⁵)₂, —N(R⁵)₂, -alkyl-COOR⁵, -alkyl-C(O)—N(R⁵)₂;        -alkyl-N(R⁵)₂; —C(O)—OR⁵; —C(O)—N(R⁵)₂; -aryl-N(R⁵)₂; acyl;        acyloxy; heterocyclo; hydroxyalkyl; —SO₂—R⁵; -alkyl-SO₂—R⁵; or        —[R³], or    -   two R groups, or an R group and an R* group, taken together with        the one or more atoms to which they are bonded, form a saturated        or unsaturated, spiro or fused, carbocyclic (such as fused        1,2-phenyl) or heterocyclic ring which may be unsubstituted or        substituted by one or more groups R or R* groups above,    -   with the proviso that a carbon atom bearing an R group is not        directly bonded to more than one heteroatom; and that at least        one R or R* is, or contains a folate-receptor binding radical        —[R³] of formula IV:    -   wherein R₀ is a folate-receptor binding residue of formula:    -    and where each X is independently —O—, —S—, —NH— or —N(R₂)—;        -   K₂ is —H, -alkyl, -alkenyl, -alkynyl, -alkoxy, -aryl,            -alkyl, —CON(R₂)₂, -glutamate, or -polyglutamate;        -   A is a linking group; and p is 0 or a positive integer;    -   R¹ is hydrogen, a thiol protecting group, or the group —R³        defined above; and    -   R₂ is independently hydrogen, alkyl, alkenyl, alkynyl, or aryl.    -   with the proviso that a carbon atom bearing an R group is not        directly bonded to more than one heteroatom;

Folate conjugates of hydrazone-containing ligands having the structuredisclosed in U.S. Pat. No. 5,651,954, incorporated herein by reference,are also useful for the preparation of metal complexes of the presentinvention.

The ligands have the following formula V:

-   -   wherein        -   Q is the group            —(C(RR))_(m1)—(Y¹)_(n)—(C(RR))_(m2)—(Y²—(C(RR))_(m3))_(n1);        -   Y¹ and Y² are each independently —CH₂—, —NR—, —O—, —S—,            —SO—, —SO₂— or —Se—;        -   n and n1 are each independently 0 or 1; and m1, m2 and m3            are independently 0 or an integer from 1 to 4; provided that            m1 and m2 are not both 0, that m1+m2+n+n1 is less than 6 and            that a carbon atom bearing an R group is not directly bonded            to more than one heteroatom;    -   each R and R* group is independently: R¹; -alkoxy; -hydroxy;        -halogen, especially fluoro; -haloalkyl; —OR¹; —C(O)—R¹;        —C(O)—N(R¹)₂; —N(R¹)₂; —N(R¹)—COR¹; -alkyl-C(O)—N(R¹)₂;        alkyl-N(R¹)₂—; -alkyl-N(R¹)—COR¹; -aryl-C(O)—OR¹;        -aryl-C(O)—N(R¹)₂; aryl-N(R¹)₂—; -aryl-N(R¹)—COR¹; -nitrile;        -acyl; -acyloxy; -heterocyclo; -hydroxyalkyl; alkoxyalkyl;        hydroxyaryl; arylalkyl; —SO₂—R¹; -alkyl-SO₂—R¹; or —R³,    -   each R¹ is independently hydrogen, alkyl, alkenyl, alkynyl or        aryl; and one to three of R, R*, or R² is, or contains a        folate-receptor binding radical —R³ of formula IV; or    -   two R groups, or an R group and an R* group, taken together with        the one or more atoms to which they are bonded, form a saturated        or unsaturated, spiro or fused, carbocyclic (such as fused        1,2-phenyl) or heterocyclic ring which may be unsubstituted or        substituted by one or more groups R or R* groups above;    -   each G¹ and G² is each independently —OH or —(NR²)₂;    -   with the proviso that at least one of G¹ or G² is —(NR²)₂, where        each R² is independently hydrogen, alkyl, aryl, acyl or —R³; and        R³ is a folate-receptor binding residue of formula IV:    -    wherein R₀ is a folate-receptor binding residue of formula:        -   each X is independently —O—, —S—, —NH— or —N(R₂)—;        -   K₂ is —H, -alkyl, -alkenyl, -alkynyl, -alkoxy, -aryl,            -alkyl, —CON(R₂)₂, -glutamate, or -polyglutamate; wherein R₂            is independently hydrogen, alkyl, or aryl;        -   A is a linking group; and p is 0 or a positive integer.

Folate-receptor binding derivatives of the following ligands arepreferred:

-   3,3,9,9-tetramethyl-4,8-diazaundecane-2,10-dione    2-(2-phenylhydrazone)10-oxime;-   3,3,9,9-tetramethyl-4,8-diazaundecane-2,10-dione    2-(2-benzoylhydrazone)10-oxime;-   3,3,9,9-tetramethyl-4,8-diazaundecane-2,10-dione-bishydrazone; and-   3,3,6,8-tetramethyl-4,8-diazaundecane-2,9-dione    2-(2-phenylhydrazone)9-oxime;    B. Methods for the Preparation of Folate Conjugates with Oxa-PnAO    Ligands    (i) Preparation of Conjugatable Oxa-PnAO Ligands

The conjugatable oxa-PnAO ligand 52b was prepared starting from theamine 50 as depicted in FIG. 18. The amine 50 was made as described byRamalingam et al. (loc. cit.). Alkylation of 50 by the chloro compound51 in the presence of diisopropylethyl amine in dimethylformamide gavethe phthalimido derivative 52a. Deprotection with hydrazine indichloromethane afforded the conjugatable amino group-bearing oxa-PnAOligand 52b.

The alkylating agent 51 was prepared as shown in FIG. 19. Potassiumphthalimidate was alkylated with 1-bromo-4-methyl-3-pentene (53a) indimethylformamide at 90° C. to obtain the phthalimido derivative 53b.Addition of isoamylnitrite to the olefin in concentrated hydrochloricacid afforded the chloronitroso compound 51.

The oxa-PnAO ligand 56 bearing two amino groups could be prepared asshown in FIG. 20. The diamine 54 has been described by Ramalingam et al.(loc. cit.). Bis-alkylation of 54 by the chloro derivative 51 isexpected to afford the bis-phthalimido derivative 55. Deprotection withhydrazine will provide the oxa-PnAO ligand 56.

ii) Preparation of Folate Conjugates with the Conjugatable Oxa-PnAOLigands

The α folate conjugate 62 of the amino group-bearing oxa-PnAO ligand 52bwas prepared as shown in FIG. 21. Coupling ligand 52b with theγ-protected glutamate derivative fMOC—NH—E(OtBu)—OH (57) using DCC/HOBTin dimethylformamide provided the glutamate derivative 58. Deprotectionwith piperidine in acetonitrile gave the amine 59.

Coupling of 59 with the pteroic acid derivative 4 using HOBT/DCCresulted in the protected conjugate 60. Deprotection with piperidinegave the product 61. Further deprotection furnished the desired folateconjugate ligand 62.

The corresponding gamma folate conjugate 64 was prepared by a similarapproach starting from the alpha protected glutamate derivativefMOC—NH—E—(OtBu) (63) as shown in FIG. 22.

Coupling ligand 52b with the oc-protected glutamate derivativefMOC—NH—E(OtBu)—OH (63) using DCC/HOBT in dimethylformamide provided theglutamate derivative 64. Deprotection with piperidine in acetonitrilegave the amine 65. Coupling of 65 with the pteroic acid derivative 4using HOBT/DCC resulted in the protected conjugate 66. Deprotection withpiperidine gave the product 67. Further deprotection furnished thedesired folate conjugate ligand 68.

iii) Preparation of Multimeric Folate Conjugates with the Oxa-PnAOLigands

Conjugates in which more than one folate residue is attached to theoxa-PnAO ligand could be useful since multiple sites of recognitioncould provide for stronger binding and higher internalization into cellsthat overexpress the folate receptor. Such ligands could be preparedfrom the oxa-PnAO ligand that bears two amino groups as in formula 56.Treatment of ligand 56 with the pteroic acid derivative 4 in thepresence of DCC/HOBT, followed by successive deprotections, first withpiperidine and then with trifluoroacetic acid is expected to furnish thebis α-folate conjugated oxa-PnAO ligand 69. The synthesis of thecorresponding bis γ-folate- or mixed bis α-, γ-folate-conjugatedoxa-PnAO analogs could be accomplished by similar methods that will beclear to those skilled in the art.

The methods provided for the folate conjugates of the oxa-PnAO ligandsare intended for preparation of compounds for use in nuclear medicineand radiotherapy applications and are based on the general oxa-PnAOligand class described in U.S. Pat. No. 5,608,110. In these ligands thefolate side chain has been attached at the oxime carbon (C═NOH) furthestfrom the oxa moiety. However, analogs wherein the folate- ormethotrexate-bearing side chain is attached at both oxime carbon atoms,as for example in formula 69, are also included in the presentinvention. Similar molecules wherein the CH₂—O—NH functionality isreplaced by CH₂—NR—NH (aza-PnAOs) are included in the present invention.The latter ligand core is covered by U.S. Pat. No. 5,651,954, which isincorporated herein by way of reference.

4. Abbreviations/Definitions

-   DMF=Dimethylformamide-   THF=Tetrahydrofuran-   DCC=Dicyclohexylcarbodiimide-   HOBT=Hydroxybenzotriazole-   TFA=Trifluoroacetic acid-   CH₃CN=Acetonitrile-   HATU=[O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium    hexafluorophosphate]-   Z=Benzyloxycarbonyl

The terms “alkyl” or “alk” as used herein alone or as part of anothergroup, denote optionally substituted, straight and branched chainsaturated hydrocarbon groups, preferably having 1 to 12 carbons in thenormal chain, most preferably lower alkyl groups. Exemplaryunsubstituted such groups include methyl, ethyl, propyl, isopropyl,n-butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl,4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl,dodecyl and the like. Exemplary substituents include one or more of thefollowing groups; halo, alkoxy, arylalkyloxy (e.g., benzyloxy),alkylthio, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl, hydroxy,carboxyl (—COOH), amino, alkylamino, dialkylamino, formyl,alkylcarbonyloxy, alkylcarbonyl, heterocyclo, aryloxy or thiol (—SH).Preferred alkyl groups are unsubstituted alkyl, haloalkyl, arylalkyl,aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, alkoxyalkyl,aryloxyalkyl, hydroxyalkyl and alkoxyalkyl groups.

The terms “lower alk” or “lower alkyl” as used herein denote suchoptionally substituted groups as described above for alkyl having 1 to 4carbon atoms in the normal chain.

The term “alkoxy” or “alkylthio” denote an alkyl group as describedabove bonded through an oxygen linkage (—O—) or a sulfur linkage (—S—),respectively. The term “alkylcarbonyl”, as used herein, denotes an alkylgroup bonded through a carbonyl group. The term “alkylcarbonyloxy”, asused herein, denotes an alkyl group bonded through a carbonyl groupwhich is, in turn, bonded through an oxygen linkage.

The term “alkenyl”, as used herein alone or as part of another group,denotes optionally substituted, straight and branched chain hydrocarbongroups containing at least one carbon to carbon double bond in thechain, and preferably having 2 to 10 carbons in the normal chain.Exemplary unsubstituted such groups include ethenyl, propenyl, butenyl,pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, and the like.Exemplary substituents include one or more alkyl groups as describedabove and/or one or more groups described above as alkyl substituents.

The term “alkynyl”, as used herein alone or as part of another groupdenotes optionally substituted, straight and branched chain hydrocarbongroups containing at least one carbon to carbon triple bond in thechain, and preferably having 2 to 10 carbons in the normal chain.Exemplary unsubstituted such groups include ethynyl, propynyl, butynyl,pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, and the like.Exemplary substituents include one or more alkyl groups as describedabove, and/or one or more groups described above as alkyl substituents.

The term “cycloalkyl”, as used herein alone or as part of another groupdenotes optionally substituted, saturated cyclic hydrocarbon ringsystems, preferably containing 1 to 3 rings and 3 to 7 carbons per ring.Exemplary unsubstituted such groups include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl,cyclododecyl, and adamantyl. Exemplary substituents include one or morealkyl groups as described above, and/or one or more groups describedabove as alkyl substituents.

The term “cycloalkenyl”, as used herein alone or as part of anothergroup, denotes such optionally substituted groups as described above forcycloalkyl, further containing at least one carbon to carbon double bondforming a partially unsaturated ring. Exemplary substituents include oneor more alkyl groups as described above, and/or one or more groupsdescribed above as alkyl substituents.

The terms “ar” or “aryl”, as used herein alone or as part of anothergroup, denote optionally substituted, homocyclic aromatic groups,preferably containing 1 or 2 rings and 6 to 12 ring carbons. Exemplaryunsubstituted such groups include phenyl, biphenyl, and naphthyl.Exemplary substituents include one or more, preferably three or fewer,nitro groups, alkyl groups as described above, and/or one or more groupsdescribed above as alkyl substituents. Preferred aryl groups areunsubstituted aryl and hydroxyaryl.

The term “carbocyclic”, as used herein alone or as part of anothergroup, denotes optionally substituted saturated, partially unsaturatedor aromatic homocyclic hydrocarbon ring systems such as the cycloalkyl,cycloalkenyl or aryl groups described above.

The terms “heterocyclo” or “heterocyclic”, as used herein alone or aspart of another group, denote optionally substituted fully saturated orunsaturated, aromatic or non-aromatic cyclic groups having at least oneheteroatom in at least one ring, preferably monocyclic or bicyclicgroups having 5 or 6 atoms in each ring. The heterocyclo group may, forexample, have 1 or 2 oxygen atoms, 1 or 2 sulfur atoms, and/or 1 to 4nitrogen atoms in the ring. Each heterocyclo group may be bonded throughany carbon or heteroatom off the ring system. Preferred groups includethose of the following formula, which may be bonded through any atom ofthe ring system:

wherein r is 0 or 1 and T is —O—, —S—, —N—R⁸ or —CH—R⁸ where R⁸ ishydrogen, alkyl arylalkyl. Exemplary heterocyclo groups include thefollowing: thienyl, furyl, pyrrolyl, pyridyl, imidazolyl, pyrrolidinyl,piperidinyl, azepinyl, indolyl, isoindolyl, quinolinyl, isoquinolinyl,benzothiazolyl, benzoxazolyl, benzimidazolyl, morpholinyl, piperazinyl,4-alkylpiperazinyl, 4-alkylpiperidinyl, 3-alkylpyrrolidinyl, oxazolyl,pyrazolyl, thiophenyl, pyridazinyl, thiazolyl, triazolyl, pyrimidinyl,1,4-dioxanyl, benzoxadiazolyl, and benzofurazanyl. Exemplarysubstituents include one or more alkyl groups as described above and/orone or more groups described above as alkyl substituents.

The terms “halogen”, “halo” or “hal”, as used here in alone or as partof another group, denote chlorine, bromine, fluorine and iodine.

The term “acyl”, as used herein alone or as part of another group,denotes the moiety formed by removal of the hydroxyl group from thegroup —COOH of an organic carboxylic acid. Exemplary such groups includealkylcarbonyl, arylcarbonyl, or carbocyclo- or heterocyclocarbonyl. Theterm “acyloxy”, as used herein alone or as part of another group,denotes an acyl group as described above bonded through an oxygenlinkage (—O—).

The term “linking group” as used herein, denotes a group which, alone ortogether with one or more other groups, covalently bonds a folatereceptor binding analog of folic acid to the remainder of a compounds ofthe present invention.

The term biomolecule as used herein, denotes a “bioactive moiety” suchas folate, which is capable of being preferentially taken up at aselected site of a subject by possessing an affinity for the folatebinding protein.

5. Detailed Description of the Methods for Metal Complexation

In preparing the compositions of the present invention, the folatereceptor-binding moiety is coupled to a metal-chelating ligand moiety,which is complexed with the metal to form a metal chelate.Alternatively, in MRI applications, the ligand may be complexed with ametal, and subsequently conjugated with the folate receptor-bindingmoiety. The ligands disclosed in (A), (B) and (C) above are complexedwith an appropriate metal for the imaging or therapeutic methodenvisioned. This may be accomplished by methodology known in the art.For example the metal can be added to water in the form of an oxide orin the form of a halide or acetate and treated with an equimolar amountof the ligand molecule. The ligand molecule can be added as an aqueoussolution or suspension. Dilute acid or base can be added (whereappropriate) to maintain a suitable pH. Heating at temperatures as highas 100° C. for period of up to 24 hours or more may sometimes beemployed to facilitate complexation, depending on the metal and thechelator, and their concentrations.

In the examples below, metal chelate synthesis will be illustrated bygadolinium (Gd) and technetium (Tc) complex synthesis. However, it is tobe understood that analogous processes can be used to prepare othermetal chelate complexes.

More particularly, the method of forming the metal complexes derivatizedwith folate according to the present invention comprises the followingsteps.

A metal complex or salt in the desired oxidation state and containing aneasily displaceable ligand or ligands (i.e., labile ligands such as H₂O,Cl, NO₃, or acetate) is mixed with the ligand of the present inventionat a pH value suitable for forming the desired complex. The labileligand is displaced from the metal by the ligands of the presentinvention to form the metal complexes of the present invention.Illustrative of such methods are the following:MX₃+LH₃→ML+3HX  (1)wherein

-   -   X is Cl, Br, F, NO₃; or acetate; and    -   M is metal such as Gd or Indium in the desired oxidation state;        MOCl₄+LH₂→MOL+2HCl+2Cl  (2a)        MO₂(R₄)^((−/0/+))+LH₂→MO₂L+4R⁻+2H⁺  (2b)        wherein    -   R is a monodentate ligand, such as pyridine, halogen, phosphine        or amine; and    -   M is a metal such as an isotope of technetium or rhenium;        MR₂+LH₂→ML+2RH+2H⁺  (3a)        MOR₂+LH₂→MOL+2RH+2H⁺  (3b)        wherein    -   R is a bidentate ligand, such as a sugar, a diol, bis amine or        bipyridine; and    -   M is a metal.

Alternatively, for radiopharmaceutical and radiotherapy applications themetal complexes of the present invention can be prepared from a metal inan oxidation state different from that of the desired complex. In thiscase, either a reducing agent or an oxidizing agent, (depending on theoxidation state of the metal used and the oxidation state of the desiredfinal product) must be added to the reaction mixture to bring the metalto the desired oxidation state. The oxidant or reductant can be used toform an intermediate complex in the desired oxidation state but withlabile ligands. These labile ligands can then be displaced by thedesired chelating ligand of the present invention. Alternatively, thelabile ligands can be added to the reaction mixture along with thereductant or oxidant and the desired ligand to achieve the change to thedesired oxidation state and chelation to the desired metal in a singlestep.

Also in accordance with the present invention, a method for diagnosticexamination or therapeutic treatment of a mammal is provided. Thismethod is based on the mechanism of receptor-mediated endocytosisactivity and involves i) the movement of a folate receptor bindingmoiety, conjugated through its alpha or gamma carboxylate to a chelatedradioactive or non-radioactive metal that can be detected by externalimaging techniques, or ii) the movement of a folate receptor bindingmoiety conjugated through its alpha carboxylate to a chemotherapy agent,into the interior of a cell through invagination of the cell membrane.The folate receptor binding moiety serves to deliver the chelated metalor a chemotherapy agent into cells that overexpress folate bindingprotein, thereby enabling diagnostic examination, radiotherapy orchemotherapeutic treatment of an organ or tissue comprising the cells.

In one aspect the method of the present invention comprises the steps of(a) administering to a mammal a composition comprising a paramagnetic orsuperparamagnetic metal complexed with a chelating ligand and coupled toa folic acid analog contained in a pharmaceutically acceptable carrierand (b) monitoring the biodistribution of the metal.

In another aspect the method of the present invention comprises thesteps of administering to a mammal a composition comprising aradioactive metal complexed with a chelating ligand coupled to a folicacid analog contained in a pharmaceutically acceptable carrier forradiotherapeutic treatment of said mammal and monitoring said treatment.

In still another aspect the method of the present invention comprisesthe steps of administering to a mammal a composition comprising achemotherapeutic agent, with or without the presence of a radioactivemetal, complexed to a folic acid analog contained in a pharmaceuticallyacceptable carrier for chemotherapy treatment of said mammal andmonitoring said treatment.

In the compositions of the present invention a folic acid analog,carrying the metal-chelate complex or the chemotherapeutic agent, bindsto folate binding protein on cell membranes, followed byinternalization.

(a) Paramagnetic Metals

Paramagnetic metals are used in affecting the relaxation times of nucleiin mammalian tissue. Certain atomic nuclei, in particular, protons,orient themselves as a result of a strong magnetic field that is appliedto them in MR imaging. The pulses of a given radio frequency, orresonance frequency, move the atomic nuclei out of a state ofequilibrium. The nuclei then return to their original state ofequilibrium as a result of spin-spin and spin-lattice relaxation. Thetime required for returning to the state of equilibrium, known asrelaxation time, gives valuable information on the degree oforganization of the atoms and on their interaction with theirenvironment.

On the basis of differences in proton density and relaxation times,images of biological tissues can be obtained which may be used fordiagnostic purposes. The greater the differences in the relaxation timesof the nuclei which are present in the tissues being examined, thegreater will be the contrast in the image that it obtained.

It is known that the relaxation times of neighboring nuclei can beaffected by the use of paramagnetic salts. In solution, the paramagneticsalts are toxic in mammals. Hence, to reduce the toxic effect ofparamagnetic metal ions administered for diagnostic purposes, they arecombined with complex compounds, i.e. complexing agents. Constituting apart of the present invention, the paramagnetic metals are complexedwith ligand moieties prior to, or subsequent to, complexation withfolates. The folate complexed with metal chelates increases theconcentration of the metal that contains high levels of FBP in cells,thus providing increased contrast of the tissue comprising the cells.

The paramagnetic metals used in the composition for MR imaging includethe elements having atomic numbers of 22 to 29, 42, 44 and 58-70.Examples of such metals are chromium (III), manganese (II), iron (II),iron (III), cobalt (II), nickel (II), copper (II), praseodymium (III),neodymium (III), samarium (III), gadolinium (III), terbium, (III),dysprosium (III), holmium (III), erbium (III) and ytterbium (III).Chromium (III), manganese (II), iron (III) and gadolinium (III) areparticularly preferred.

Doses for administration of paramagnetic metals in the complex of thepresent invention are from about 0.05 to about 0.3 mmol/kg of bodyweight.

The metal complexes of the present invention find utility as diagnosticand/or therapeutic agents. Thus, the present invention provides methodsfor the diagnosis of the presence and/or status of a disease state, orfor the treatment of a disease state, comprising the step ofadministering a metal complex of the present invention to a subject inneed thereof. The metal complexes of the present invention may beadministered by an appropriate route such as orally, parentally (forexample, intravenously), intramuscularly or intraperitoneally or by anyother suitable method. For example, the complexes of this invention maybe administered to a subject by bolus or slow infusion intravenousinjection.

(b) Radioactive Metals

In the embodiment of the present invention directed to radiographicimaging or radiotherapy, radioisotopes are utilized. Preferredradioisotopes include: ^(99m)Tc, ⁵¹Cr, ⁶⁷Ga, ⁶⁸Ga, ¹¹¹In, ¹⁶⁸Yb, ¹⁴⁰La,⁹⁰Y, ⁸⁸Y, ¹⁵³Sm, ¹⁵⁶Ho, ¹⁶⁵Dy, ⁶⁴Cu, ⁹⁷Ru, ¹⁰³Ru, ¹⁸⁶Re, ¹⁸⁸Re, ²⁰³Pb,²¹¹Bi, ²¹²Bi, ²¹³Bi and ²¹⁴Bi. The choice of metal ion will bedetermined based on the desired therapeutic or diagnostic application.

The amount of radiopharmaceutical administered may be selected based onthe desired use, such as to produce a diagnostic image of an organ, bymethods known in the art. Doses may range from about 2 to 200 mCi, or aslimited by the in vivo dosimetry provided by the radiopharmaceuticals.The radiopharmaceutical may optionally be co-administered with ametal-free ligand of the folic acid derivatives of the present inventionwhich derivative is present in an amount of from about 0.05 mg to about200 mg per dose.

6. General Description of the Conjugation of Folates withChemotherapeutic Agents

In this embodiment the present invention comprises a chemotherapeuticcompound complexed with a folate receptor-binding ligand through itsalpha carboxylate or its alpha and gamma carboxylate functionality,which on administration to a patient, is capable of selectivelyenhancing the transport of the chemotherapeutic agent across themembrane of cancer cells that overexpress FBP and decreasing the uptaketo non-target organs, thereby facilitating treatment of the tumor beingtargeted.

Chemotherapeutic agents useful in neoplastic disease are listed, forexample, in Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 6^(th) Ed., 1980, MacMillan Publ. Co., N.Y., pp.1252-1254, The Merck Index, 11^(th) Ed. 1989, which are incorporatedherein by reference. These chemotherapeutic agents include:

Alkylating Agents

-   -   Alkyl Sulfonates, such as        -   Busulfan,        -   Improsulfan, and        -   Piposulfan,    -   Aziridines, such as        -   Benzodepa,        -   Carboquone,        -   Meturedepa, and        -   Uredepa    -   Ethylenimines and Methylmelamines        -   Altretamine,        -   Triethylenemelamine,        -   Triethylenephosphoramide,        -   Triethylenethiophosphoramide, and        -   Trimethylolmelamine    -   Nitrogen Mustards, such as        -   Chlorambucil,        -   Chlomaphazine,        -   Cyclophosphamide,        -   Estramustine,        -   Ifosfamide,        -   Mechlorethamine,        -   Mechlorethamine Oxide Hydrochloride,        -   Melphalan,        -   Novembichin,        -   Phenesterine,        -   Prednimustine,        -   Trofosfamide and        -   Uracil Mustard    -   Nitrosoureas, such as        -   Carmustine,        -   Chlorozotocin,        -   Fotemustine,        -   Lomustine,        -   Nimustine, and        -   Ranimustine    -   Antibiotics, such as        -   Aclacinomycins,        -   Actinomycin F₁,        -   Anthramycin,        -   Azaserine,        -   Bleomycins,        -   Cactinomycin,        -   Carubicin,        -   Carzinophilin,        -   Chromomycins,        -   Dactinomycin,        -   Daunorubicin,        -   Doxorubicin,        -   Epirubicin,        -   Mitomycins,        -   Mycophenolic Acid,        -   Nogalamycin,        -   Olivomycins,        -   Peplomycin,        -   Plicamycin,        -   Porfiromycin,        -   Puromycin,        -   Streptonigrin,        -   Streptozocin,        -   Tubercidin,        -   Ubenimex,        -   Zinostatin, and        -   Zorbucin    -   Antimetabolites, such as        -   Fludarabine,        -   6-Mercaptopurine,        -   Thiamiprine,        -   Thioguanine,        -   Ancitabine,        -   Azacitidine,        -   6-Azauridine,        -   Camofur,        -   Cytarabine,        -   Doxifluridine,        -   Enocitabine,        -   Floxuridine,        -   Fluorouracil,        -   Tegafur, and        -   L-Asparaginase            Antineoplastic (Hormonal)    -   Androgens, such as        -   Calusterone        -   Dromostanolone Propionate,        -   Epitiostanol,        -   Mepitiostane and        -   Testolactone    -   Antiadrenals, such as        -   Aminoflutethimide,        -   Mitotane, and        -   Trilostane    -   Antiandrogens, such as        -   Flutamide and        -   Nilutamide    -   Antiestrogens, such as        -   Tamoxifen and        -   Toremifene    -   Estrogens, such as        -   Fosfestrol,        -   Hexestrol and        -   Polyestradiol Phosphate    -   LH-RH Analogs, such as        -   Buserelin,        -   Goserelin,        -   Leuprolide and        -   Triptorelin    -   Progestogens, such as        -   Chlormadinone Acetate,        -   Medroxyprogesterone        -   Megestrol Acetate, and        -   Melengestrol

The therapeutic complexes of this invention may be administered to amammal alone or in combination with pharmaceutically acceptablecarriers, the proportion of which is determined by the chemical natureof the chemotherapeutic compound, chosen route of administration andstandard pharmaceutical practice.

The physician will determine the most suitable dosage of the presenttherapeutic agents and it will vary with the form of administration andthe particular compound chosen, and also, it will vary with theparticular patient under treatment. Dose levels will be equal to orlower than those used with the chemotherapeutic compounds alone sincethe folate complex effectively delivers the chemotherapeutic compoundinto the tumor cells.

In chemotherapy the complexes of the present invention can beadministered through intravenous, intramuscular or intraperitonal routesin a physiologically acceptable medium, such as saline or water that isbuffered or pH adjusted using physiologically acceptable salts orbuffers well-known in the art.

The complexes can be administered as a bolus by continuous infusion orgiven on alternative days determined by experimental methods which arewell known to skilled chemotherapists.

7. Description of Kits for Forming Metal Complexes

For radiopharmaceutical or radiotherapy applications it is convenient toprepare the complexes of the present invention at, or near, the sitewhere they are to be used. A single, or multi-vial kit that contains allof the components needed to prepare the complexes of this invention,other than the radionuclide ion itself, is an integral part of thisinvention.

The amount administered may be selected based on the desired use, suchas to produce a diagnostic image of an organ or other site of a subjector a desired radiotherapeutic effect, by methods known in the art.Exemplary dosages are those employing about 2-200 mCi rhenium (forradiotherapy) or about 10-60 mCi technetium (for imaging). The “subject”of the methods of the present invention is preferably a mammal such as adomestic mammal, for example, a dog, cat, horse or the like, or mostpreferably, a human. Depending upon the metal and ligand used, thecomplexes of the present invention may be employed as, for example,imaging agents useful for imaging tissues or organs that overexpressfolate binding protein, such as tumor cells, epithelial cells, kidneys,gastrointestinal or the hepatobiliary system.

Preferred complexes of the present invention are those comprising aligand complexed with a radionuclide such as technetium, or rhenium.

Rhenium is particularly useful as a radiotherapy agent. The rheniumemployed is preferably one of the radionuclides Re-186 or Re-188, or amixture thereof, which mixture may also include Re-185 and/or Re-187.Preparation of the complexes of the present invention where the metal isrhenium may be accomplished using rhenium in the +5 or +7 oxidationstate. Examples of compounds in which rhenium is in the Re(VII) stateare NH₄ReO₄ or KReO₄. Re(V) is available as, for example, [ReOCl₄](NBu₄), [ReOCl₄] (AsPh₄), ReOCl₃ (PPh₃) and as ReO₂ (pyridine)₄+. (Ph isphenyl; Bu is n-butyl). Other rhenium reagents capable of forming arhenium complex may also be used.

Technetium is particularly useful as a diagnostic imaging agent. Thetechnetium employed is preferably one or more of the radionuclidesTc-99m, Tc-94m or Tc-96. The preferred radioisotope for medical imagingis ^(99m)Tc. Its 140 keV γ-photon is ideal for use with widely-availablegamma cameras. It has short (6 hour) half like, which is desirable whenconsidering patient dosimetry. ^(99m)Tc is readily available atrelatively low cost through commercially-produced ⁹⁹Mo/^(99m)Tcgenerator systems. Preparation of the complexes of this invention wherethe metal is technetium may be accomplished using technetium in the formof the pertechnetate ion. For Tc-99m, the pertechnetate ion ispreferably obtained from commercially available technetium-99mparent-daughter generators; such as technetium is in the +7 oxidationstate. The generation of the pertechnetate ion using this type ofgenerator is well known in the art, and is described in more detail inU.S. Pat. Nos. 3,369,121 and 3,920,995. These generators may generallybe eluted with saline solution, and the pertechnetate ion obtained asthe sodium salt. Pertechnetate may also be prepared fromcyclotron-produced radioactive technetium using procedures well know inthe art.

A preferred single-vial kit of the present invention comprises a liganddescribed in sections A, B or C, a folic acid derivative of the α-, γ-and bis isomers in a desired ratio, and a source of a pharmaceuticallyacceptable reducing agent such as a stannous salt. More preferably, inaddition, the kit is buffered with a pharmaceutically acceptable acid orbase to adjust the pH to a desired value for complex formation. It ispreferred that the kit contents be in lyophilized form. Such a singlevial kit may optionally contain exchange ligands such as glucoheptonate,gluconate, mannitol, malate, citric or tartaric acid and may alsocontain reaction modifiers, such as diethylenetriaminepentaacetic acidor ethylenediamine tetraacetic acid. Additional additives, such assolubilizers (for example α-, β- or γ-cyclodextrin), antioxidants (forexample ascorbic acid) and/or fillers (for example, NaCl) may beemployed to improve the radiochemical purity and stability of the finalproduct, or to aid in the production of the kit.

A preferred multi-vial kit of the present invention comprises, in onevial, the components, other than the radionuclide itself, required toform a labile radionuclide (especially Tc(V)) complex, that is, anexchange ligand and a pharmaceutically acceptable reducing agent such asa stannous salt. The quantity and type of exchange ligand, and amountand type of reducing agent and buffer used may be selected based on thenature of the exchange complex to be formed. The ligand described in A,B, C, D, or E, a folic acid derivative of the α, γ and bis isomers in adesired ratio of the present invention is contained in a second vial, aswell as optional additives such as buffers appropriate to adjust the pHto its optimal value.

A single vial kit may be ready for use following addition of theradionuclide ion, such as pertechnetate. A multi-vial kit may be readyfor use by addition of the radionuclide ion, such as pertechnetate, tothe vial containing exchange ligand and reducing agent, and afterwaiting an appropriate period of time for formation of a labile complex,the contents of this vial are added to the second vial containing asource of the desired ligand. After a reaction time of about 1 to 60minutes, the complex of the present invention is formed. It isadvantageous that the contents of both vials of this multi-vial kit belyophilized. As described for the single vial kit, additional additivesmay be employed to improve the radiochemical purity and stability of thefinal product, or to aid in the production of the kit.

Alternatively, the multi-vial kit may comprise the desired ligand in onevial and a source of reducing agent such as stannous ion in a secondvial. Pertechnetate may be added to the vial containing ligand, and thenthe contents of the second vial added to initiate labeling. As above,the quantity and type of ligand, buffer pH and reducing agent may beselected based on the nature of the desired ligand use. Again, it isadvantageous that the contents of both vials be lyophilized.

8. Examples

A. Synthesis of Intermediates and Folate Conjugates

EXAMPLE 1 N-Pteroyl-γ-glutamyl-APADO3A (6) [DO3A-APA-(γ)-folate]

A) N-CBZ-α-t-butyl-L-glutamyl-APADO3A-tri-t-butyl ester (3)

To a cooled solution of N-CBZ-L-glutamic acid-α-t-butyl ester (1) (3.75g; 11.1 mmol) in DMF (30.0 mL) were added HATU (5.25 g, 13.8 mmol) anddiisopropylethylamine 4.45 g (34.4 mmol) and the mixture was stirred atRT for 15 min. APADO3A-tris-t-butyl ester (2) 6.0 g (9.1 mmol) was addedand the reaction mixture was stirred at RT for 12 h. DMF was removedunder vacuum and the residue was treated with water and extracted withethyl acetate. The organic layer was washed with 10% NaOH (3×100 mL),water and dried (Na₂SO₄). Ethyl acetate was removed on a rotaryevaporator and the thick oil obtained was chromatographed over silicagel (CH₂Cl₂:CH₃OH, 95:5). UV visible fractions were collected and thesolvent was removed to give a viscous oil which was dried under vacuumto obtain a foamy solid. Yield 5.5 g (93%). MS: (M+H)⁺=982.7.

B) L-glutamyl-α-t-butyl-APADO3A-tri-t-butyl ester (3a)

To a solution of N-CBZ-α-t-butyl-L-glutamyl-APADO3A-tri-t-butyl ester(3) (1.0 g; 4.6 mmol) in methanol (50.0 mL) was added 5% Pd-C (500 mg)and the mixture was hydrogenated (30 psi) for 12 h. Catalyst was removedby filtration and methanol was removed to give a colorless thick oil. Itwas dried under vacuum to afford a foamy solid. Yield 0.82 g (98%). MS:(M+Na)⁺=871. HRMS (FAB) m/z, Calcd for C₄₃H₇₃N₇O₁₀ (M+Na⁺) 871.5395;Found: 871.5325.

C)N-(N¹⁰-trifluoroacetylpteroyl)-α-t-butyl-L-glutamyl-APADO3A-tris-t-butylester (5)

To a stirred, 0° C. slurry of N¹⁰-trifluoroacetylpteroic acid (4) (0.204g; 0.5 mmol) in dimethylformamide [DMF] was added hydroxybenzotriazole(0.092 g, 0.6 mmol). After 10 min dicyclohexylcarbodiimide [DCC] (0.125g, 0.6 mmol) was added and the slurry was stirred at 0° C. for 1 h. Tothis suspension was added L-glutamyl-α-t-butyl-APADO3A-tri-t-butyl ester(3a) (0.45 g; 0.53 mmol) followed by diisopropylethylamine (0.13 g, 1mmol). The reaction mixture was allowed to stir for 2 h at 0° C. andthen 12 h at RT. DMF was removed under reduced pressure and the residuewas treated with water. The light yellow solid formed was filtered anddried under vacuum. Trituration of the solid with hot ethyl acetate(3×50 mL) and removal of the solvent yielded a light yellow solid. Theproduct was purified by silica gel column chromatography, eluting withCH₂Cl₂:CH₃OH (95:5), to afford 0.25 g (40%) of the amide as a whitesolid. MS: (M+H)⁺=1238.5. (M+Na)⁺=1260.4.

D) N-Pteroyl-α-t-butyl-L-glutamyl-APADO3A-tris-t-butyl ester (5a)

To a solution ofN-(N¹⁰-trifluoroacetylpteroyl)-α-t-butyl-L-glutamyl-APADO3A-tris-t-butylester (5) (0.31 g; 0.25 mmol) in DMF-water (4.5:0.5, 5 mL), piperidine(0.3 mL) was added and the solution was stirred at RT for 24 h.DMF-water were removed under vacuum to give a thick oil. The oil wastreated with water (5 mL) and the precipitated yellow solid wasfiltered, dried under vacuum. Purification by silica gel columnchromatography (CH₂Cl₂:CH₃OH, 95:5) yielded 0.47 g (72.0%). ofN-Pteroyl-α-t-butyl-L-glutamyl-APADO3A-tris-t-butyl ester.

This compound was further purified by reverse phase HPLC (Vydac-C18,10μ, 10×25 cm) with a linear gradient of 0.1% TFA in H₂O/CH₃CN (0-60%)over sixty min to give 0.2 g of the product. MS: (M+H)⁺=1143. HRMS (FAB)m/z, Calcd for C₅₇H₈₃N₁₃O₁₂ (MH⁺) 1142.6379; Found: 1142.6362.

E) N-Pteroyl-γ-L-glutamyl-APADO3A (6)

N-Pteroyl-α-t-butyl-L-glutamyl-APADO3A-tris-t-butyl ester (5a) (0.16 g;0.14 mmol), was dissolved in concentrated hydrochloric acid (0.3 mL) andstirred for 15 min. Absolute ethanol (3.0 mL) was added to the reactionmixture and the precipitated hydrochloride was centrifuged and thesupernatant solution was decanted. The hydrochloride was suspended inethanol (3.0 mL), stirred for 5 min, centrifuged, and decanted. Thesolid was treated in the same manner with two additional volumes ofethanol (3.0 mL ). The light yellow hydrochloride obtained was driedunder vacuum. Yield 0.16 g. MS: (M+H)⁺=918.

EXAMPLE 2 Gd-DO3A-APA-(γ)-folate

DO3A-APA-(γ)-folate ligand (6) (74 mg; 0.0685 mmoles) was suspended in20 mL of water and adjusted to pH 5.5 with 26.5 mL of 0.01M NaOH.GdCl₃.4H₂O (0.0716 mmoles) in 1.79 mL of 0.04M HCl was added and thesolution was stirred under nitrogen at ˜40° C. as the reaction mixturepH was gradually adjusted to pH 5.5 using 0.01N NaOH. After four hours,ethanol (50 mL) was added and the reaction mixture volume was reduced to40 mL by rotary evaporation. Addition of ethanol (80 mL) caused theprecipitation of an orange gel, which was isolated by centrifugation at4° C. The pellet was rinsed with 20 mL of ethanol, and dried in vacuo toyield 35 mg of product. Anal. Calcd. for the Na salt ofGd-DO3A-APA-(γ)-folate 3H₂O.2EtOH.0.15NaCl (C₄₅H₆₆N₁₃O₁₇GdNa.0.15NaCl,MW=1253.04): Calcd: C, 43.13; H, 5.31; N, 14.53; Cl, 0.566. Found: C,43.21; H, 4.96; N, 14.47; Cl, 0.56.

Alternatively, the isolated gel was dissolved in 10 mM (NH₄)HCO₃ andchromatographed on a DEAE Tris-acryl anion exchange column. Traceimpurities were removed from the column by elution with 10 mM (NH₄)HCO₃(200 mL). Product was then eluted from the column using a gradient of 10mM to 100 mM ammonium bicarbonate, and fractions containing the compoundwere freeze-dried to give product that is consistent with the anhydrousformulation Na(Gd-DO3A-APA (γ) folate.CO₃. Anal Calcd. forC₄₂H₄₉N₁₃O₁₅GdNa, (mw=1156.19): Calc. C, 43.63; H, 4.27; N, 15.75.Found: C, 43.84; H, 4.10; N, 15.76.

EXAMPLE 3 ¹⁵³Gd-DO3A-APA-(γ)-folate

DO3A-APA-(γ)-folate ligand.3HCl.3H₂O (6) (10.13 mg, 0.00936 mmoles) wasmixed with 7.5 mL of distilled water and adjusted to pH 5.86. A 1 MLaliquot of this solution (0.863 mg, 0.7975 μmol) was added to a 2-dramvial. A solution of GdCl₃.4H₂O (1.34 mg/mL) was prepared in dilute HCl.An 0.2 mL aliquot of this solution was mixed with 100 μCi of ¹⁵³GdCl₃ in0.5M HCl and then transferred to the ligand vial. The resulting solutionwas heated at 55° C. with stirring, and the pH was adjusted to 6.0 over30 minutes using 0.1N NaOH. After 1.5 hours, the pH had fallen to 5.2and ¹⁵³Gd-DO3A-APA-(γ)-folate had formed in 96% yield as determined byradioisotope detection using HPLC on a Supelcosil LC-18 column (25cm×4.6 mm) eluted with a step gradient of 0 to 40% acetonitrile/bufferA. (buffer A=1.0 mM tris buffer pH 7.25 containing 0.2 mM EDTA).

EXAMPLE 4 N-Pteroyl-α-L-glutamyl-APADO3A (10) [DO3A-APA-(α)-folate]

A) N-CBZ-γ-t-butyl-L-glutamyl-APADO3A-tri-t-butyl ester (9)

To an ice-cooled, stirred solution of N-CBZ-L-glutamic acid γ-t-butylester (7) (1.2 g; (3.7 mmol) and HATU (1.75 g, 4.6 mmol) in DMF (15 nmL)was added diisopropyl ethylarnine (1.48 g , 11.4 mmol) under nitrogen.The mixture was stirred at 0° C. for 15 min. APADO3A-tris-t-butylester(2) (2.0 g; 3.0 mmol) was added and the reaction mixture was stirred atRT for 12 h. DMF was removed under vacuum and the residue was treatedwith water and extracted with ethyl acetate (2×150 mL). The ethylacetate layer was washed with NaOH (10%), water and dried (Na₂SO₄). Thesolvent was removed on a rotary evaporator and the residue was purifiedby column chromatography on silica gel with 5% methanol in CH₂Cl₂ as theeluant. UV visible fractions were collected and methylene chloride andmethanol were removed to give a viscous oil which was dried under vacuumto give a foamy solid. Yield: 2.42 g (82%). MS: (M+H)⁺=982.7.

B) L-glutamyl-γ-t-butyl-APADO3A-tri-t-butyl ester (8a)

N-CBZ-γ-t-Butyl-L-glutamyl-APADO3A-tris-t-butylester (8) (2.0 g; 2.0mmol) was hydrogenated (50 psi) in methanol (50.0 mL) over 30% Pd-C (200mg) at RT for 12 hours. The catalyst was removed by filtration on Celiteand washed with methanol (3×10 mL). The combined methanolic solution wasevaporated to a thick viscous oil. It was dried under vacuum to afford1.6 g (92%) of L-glutamyl-γ-t-Butyl-APADO3A-tris-t-butylester 8a as afoamy solid. MS: (M+Na)⁺=871. HRMS (FAB) m/z, Calcd for C₄₃H₇₃N₇O₁₀(M+Na⁺⁾ 871.5395; Found: 871.5414.

C)N-(N¹⁰-trifluoroacetylpteroyl)-γ-t-butyl-L-glutamyl-APADO3A-tris-t-butylester (9)

To a stirred, 0° C. slurry of N¹⁰-trifluoroacetylpteroic acid (4) (0.204g, 0.5 mmol) in DMF was added DCC (0.125 g, 0.6 mmol) and the slurry wasstirred at 0° C. for 1 h. To this suspension was addedL-glutamyl-γ-t-butyl-APADO3A-tri-t-butyl ester 8a (0.45 g, 0.53 mmol)followed by diisopropylethylamine (0.13 g, 1 mmol). The reaction mixturewas allowed to stir for 2 h at 0° C. and then 12 h at RT. DMF wasremoved under reduced pressure and the residue was treated with water.The light yellow solid formed was filtered and dried under vacuum.Trituration of the solid with hot ethyl acetate (3×50 ml) and removal ofthe solvent yielded a light yellow solid. The product was purified bysilica gel column chromatography, eluting with CH₂Cl₂:CH₃OH (95:5), toafford 0.185 g (30%) of the amide as a white solid. MS: (M+H)⁺=1238.5.HRMS (FAB) m/z, Calcd for C₅₉H₈₂N₁₃O₁₃F₃ (M+Na⁺) 1260.6005; Found:1260.6033.

(D) N-Pteroyl-γ-t-butyl-L-glutamyl-APADO3A-tris-t-butyl ester (9a)

To a solution of APADO3A folate (9) (0.16 g; 125 mmol) in DMF-water(4.5:0.5 mL), piperidine (0.2 mL) was added and the solution was stirredat RT for 24 h. DMF-water were removed under vacuum and the residue wastreated with water (5 mL). The precipitated yellow solid was filtered,dried under vacuum and purified by silica gel column chromatography(CH₂Cl₂:CH₃OH, 95:5). UV visible fractions were collected and methylenechloride and methanol were removed to give a solid. Yield: 95 mg(72.0%). The compound was further purified by reverse phase HPLC(Vydac-C18, 10μ, 10×25 cm) with a linear gradient of 0.1% TFA inH₂O/CH₃CN (0-60%) over sixty min to give 55 mg of yellow solid. MS:(M+H)⁺=1143. HRMS (FAB) m/z, Calcd for C₅₇H₈₃N₁₃O₁₂ (MH⁺) 1142.6362;Found: 1142.6426.

E) N-PteroyI-γ-L-glutamyl-APADO3A (10).

N-Pteroyl-α-t-butyl-L-glutamyl-APADO3A-tris-t-butyl ester (9a) (0.1 g,0.087 mmol), was treated with concentrated hydrochloric acid (0.2 mL)and the mixture was stirred for 15 min. Absolute ethanol (3.0 mL) wasadded to the reaction mixture and the precipitated solid was centrifugedand the supernatant solution was decanted. The solid was triturated withethanol (3.0 mL), centrifuged and decanted. The solid was treated in thesame manner with two additional volumes of ethanol (3.0 mL) and thehydrochloride obtained was dried under vacuum. Yield 82 mg. MS:(M+H)⁺=918.

EXAMPLE 5 Gd-DO3A-APA-(α)-folate

DO3A-APA-(α)-folate.4HCl.4H₂O (10) (137 mg, 0.1206 mmoles) was suspendedin 4 mL of 0.05M NaOH with stirring under N₂. Solid GdCl_(3.)4H₂O (45.3mg, 0.135 mmol) was added to the orange gel-like suspension and NaOH(0.05M) was gradually added dropwise to maintain a pH of ˜4.5 to 5.0.After 24 hours, the yield of product was 81.4% as determined using HPLC(A₂₇₄). Water (5 mL) was added, and the pH of the solution was adjustedto 7.0 with 0.05M NaOH. The slightly hazy bright-yellow solution wasfiltered into a centrifuge tube, an equal volume of acetone (15 mL) wasadded, and the mixture was chilled to −20° C. The resulting orangeprecipitate was isolated by centrifugation at 4° C., rinsed withice-cold ethanol, re-dissolved in a minimal volume of water andfreeze-dried to yield 113.8 mg of pale yellow Gd-DO3A-APA-((α)-folateproduct (77.2% yield), as determined by HPLC on a Supelcosil LC-18column (25 cm×4.6 mm) eluted with a step gradient of 0 to 40%acetonitrile/buffer A. (buffer A=1.0 mM tris buffer pH 7.25 containing0.2 mM EDTA). MS: (M+H)⁺=1072; (M+H+H₂O)⁺=1090. Anal. Calcd. forC₄₁H₄₇N₁₃O₁₂NaGd 5.5 H₂O 0.5 NaCl: C, 40.28; H, 4.78; N, 14.90, Na,2.82, H₂O, 8.11. Found: C, 40.41; H, 4.34; N, 14.57, Na, 2.71, H₂O,8.07.

EXAMPLE 6 ¹⁵³Gd-DO3A-APA-(α)-folate

DO3A-APA-(α)-folate 4HCl.4H₂O (10) (8.33 mg, 0.00733 mmoles) was mixedwith 5.0 mL of distilled water and adjusted to pH 6.8 with 0.1N NaOH. A25.3 μL aliquot of this solution (35.7 nmol) was added to a 2-dram vial.To this was added 100 μCi of ¹⁵³GdCl₃ in 0.5M HCl (35.7 nmol, 25.3 μL)and the solution was adjusted gradually to pH 5.2 using 0.1 N NaOH.After the reaction was stirred at room temperature for 3 days,¹⁵³Gd-DO3A-APA-(α)-folate had formed in 51% yield as determined byradioisotope detection using HPLC on a Supelcosil LC-18 column (25cm×4.6 mm) eluted with a step gradient of 0 to 40% acetonitrile/bufferA. (buffer A=1.0 mM tris buffer pH 7.25 containing 0.2 mM EDTA).

EXAMPLE 7 N-Pteroyl-L-glutamyl-bis APADO3A (14) [Bis[DO3A-APA]-folate]

A) N-CBZ-L-glutamyl-bis APADO3A-tris-t-butyl ester (14)

To a cooled (0° C.) solution of N-CBZ-glutamic acid (11) (0.28 g, 1.0mmol) in dimethyl formamide [DMF] (5.0 mL) were added HATU (1.0 g, 2.63mmol) and diisopropyl ethylamine (0.52 g, 1.4 mL, 3.85 mmol) and themixture was stirred at 0° C. for 15 min. APADO3A tris-t-butyl ester (2)(1.32 g, 2.0 mmol) was added to the reaction mixture and stirred at 0°C. for 1 h and at RT for 12 h. Potassium carbonate (1.0 g) was added tothe reaction mixture, which was then stirred for 10 min. DMF was removedunder vacuum and the residue was treated with water and dried. Crudeyield: 1.52 g.

The crude coupled product was then purified by silica gel columnchromatography (CH2Cl₂: CH₃OH, 95:5). Product-containing fractions werecollected and the purity was determined by HPLC. Yield: 0.98 g (62%).MS: (M+Na)⁺=1593.

B) L-glutamyl-bis APADO3A-tris-t-butyl ester (12a)

CBZ-derivative (12) (0.5 g, 0.3 mmol) was dissolved in methanol (10 mL)and hydrogenated at 40 psi using 5% Pd-C (degussa type) for 12 h. Thecatalyst was removed by filtration and the methanol was removed on arotary evaporator to give an oil. It was dried under vacuum to give afoamy solid. Yield: 0.45 g (98%). MS: (M+Na)⁺=1459.

C) N-(N¹⁰-trifluoroacetylpteroyl)-L-glutamyl-bisAPADO3A-tris-t-butylester (13)

To a cooled solution (0° C.) of N¹⁰-trifluoroacetylpteroic acid (4)(0.13 g, 0.32 mmol) in dimethylformamide [DMF] (4.0 mL) was addedhydroxybenzotriazole (0.052 mg, 0.34 mmol) and the mixture was stirredat 0° C. for 15 min. Cyclohexyl carbodiimide (0.08 g) was added to thereaction mixture and the mixture was stirred at 0° C. for 1 h.L-glutamyl-bis APA DO3A-tris-t-butyl ester (12a) was then added to thereaction mixture followed by diisopropylethylamine and stirred at RT for18 h. The reaction mixture was concentrated under vacuum and the residuewas treated with water. The solid obtained was filtered and dried undervacuum. The crude compound was triturated with ethyl acetate anddecanted. The crude compound was chromatographed over silica gel usingmethylene chloride and methanol as the eluent. Fractions containing thecompound were collected and evaporated to give 0.28 g of the coupledproduct. This was then treated with 0.25 g of decolorizing carbon inmethanol. The carbon was filtered and methanol was removed to give 0.25g of the pure product. MS: (M+2Na)⁺1872.

D) N-Pteroyl-L-glutamyl-bisAPADO3A-tris-t-butyl ester (13a)

To a solution of the trifluoroacetyl derivative (13) (0.17 g; 0.099mmol) in DMF:water (4.5: 0.5 mL) was added piperidine (0.3 mL) and themixture was stirred at RT for 24 h. DMF was removed under vacuum and theresidue was treated with water. The solid obtained was dissolved inmethanol (5.0 mL) and treated with decolorizing carbon (100 mg). Thecarbon was removed by filtration and the methanolic solution wasconcentrated to give yellow solid. Yield: 140 mg. MS: (M +Na)⁺=1753.

E) N-Pteroyl-L-glutamyl-bis-APADO3A (14)

N-Pteroyl-L-glutamyl-bisAPADO3A-tris-t-butyl ester (13a) (0.25 g, 0.145mmol), was treated with concentrated hydrochloric acid (0.2 mL) and themixture was stirred for 15 min. Absolute ethanol (3.0 mL) was added tothe reaction mixture and the precipitated solid was centrifuged and thesupernatant solution was decanted. The solid was triturated with ethanol(3.0 mL), centrifuged and decanted. The solid was treated in the samemanner with two additional volumes of ethanol (3.0 mL) and thehydrochloride obtained was dried under vacuum. Yield 150 mg. MS:(M+H)⁺=1395.

EXAMPLE 8 Bis (Gd-DO3A-APA)-folate

Bis(DO3A-APA)-folate.4HCl.10H₂O ligand (14) (75 mg, 0.0414 mmol) wasdissolved in 1 mL of water and a solution of GdCl₃ in H₂O (0.095 mmol,190 μL) was added with stirring. The pH of the reaction mixture wasgradually raised to 6.8 with 1N NaOH, and the solution was allowed tostir overnight at RT in the dark. At 18 hours additional GdCl₃ in H₂O(0.003 mmol, 5.7 μL) was added. At 24 hours complex formation wasdetermined to be 90% using HPLC (Supelcosil C-18-DB column, 0 to 40%acetonitrile/buffer A step gradient, buffer A=1.0 mM tris acetate bufferpH 7.25, containing 0.2 mM EDTA). Purification was accomplished usingsemi-preparative HPLC (Supelcosil C-18-DB column, 0 to 40%acetonitrile/water step gradient). The complex cuts were pooled andlyophilized and 36 mg of purified material obtained. After freezedrying, the HPLC purity was 97.3% at A₂₇₄. Overall yield: 50%.Analytical data, calculated for the hydrated HCl salt ofBis-(Gd-DO3A-APA-)folate.2HCl.14H₂O (Gd₂C₆₃O₁₈N₁₉H₇₆(2HCl.14H₂O)Mw=2027.7): Calculated C, 37.32%; H, 5.27%; N, 13.12%. Found C, 37.37%;H, 5.04%; N, 12.36%. MS (Electrospray): (M+H)⁺, m/z=1701; (M+2H)²⁺ Gd₂cluster, m/z=852 [base peak].

EXAMPLE 9 Bis (¹⁵³Gd-DO3A-APA)-folate

A ligand stock solution (1 mg/mL, 557 nmol/mL) was prepared bydissolving 10 mg of bis(DO3A-APA)-folate 4HCl.10H₂O ligand (14) in 10 mLof water. A carrier-added reaction mixture with a 1:1 metal to ligandratio was prepared by premixing ¹⁵³GdCl₃/0.5M HCl (23.2 μL, 100 μCi,62.09 nmol Gd) with an aliquot of a 10 mM GdCl₃ stock solution (55.9 μL,557 nmol), in a 1-dram bottle with stirring, followed by an aliquot ofthe bis(DO3A-APA)-folate ligand stock solution (557 μL, 310 nmol). ThepH of the reaction mixture was gradually raised to 4.9 over two hourswith dilute NaOH and it was then stirred in the dark overnight at RT. At18 hours the pH was raised to 5.8. Complexation was monitored using HPLC(Supelcosil C18 column, acetonitrile/buffer A step gradient, bufferA=1.0 mM tris buffer pH 7.25 containing 0.2 mM EDTA). At 48 hours the pHwas 5.8, and complex yield was 65%. The solution was reduced in volumeto 250 μL using a nitrogen stream, the compound was purified usingpreparative HPLC (Supelcosil C18, acetonitrile/water step gradient), andthe ¹⁵³Gd/Gd-bis-complex-containing fractions were evaporated to drynessunder a nitrogen stream. The material was reconstituted in 1.0 mL ofwater and the pH adjusted to 8.0 using 20 mM Tris HCl buffer. Overallyield: 41% as determined from recovered radioactivity. Radiochemicalpurity (RCP) was determined by HPLC as 96.4%.

EXAMPLE 10 Tris-t-butylN-12-(3-amino-2-methoxycarbonyl-1-ethyl)-1,4,7,10-tetracyclododecane-1,4,7-tricarboxylate(17a)

A) Methyl 3-amino-2-hydroxypropionate hydrochloride (19)

A suspension of isoserine (18) (20.0 g, 0.19 mol) in anhydrous methanol(100 mL) was saturated with HCl gas at 0° C. while being stirred withexclusion of moisture. The solid material slowly dissolved to yield apale yellow liquid after a few minutes. The HCl saturated solution wasstirred overnight (20 h) at room temperature. Excess HCl gas was removedby bubbling nitrogen into the reaction mixture and the solvent wasremoved under reduced pressure to yield the product as a syrup (27.1 g;yield 92%) Mass Spectrum M+H 120. The product was immediately taken tothe next step without further purification.

B) Methyl 3-azido-2-hydroxypropionate

i) Preparation of triflylazide

To an ice-cold solution of sodium azide (166.0 g, 2.6 mol) in water (500mL) dichloromethane (300 mL) was added, followed bytrifluoromethanesulfonic anhydride (144.0 g, 0.51 mol) dropwise whilemaintaining the solution at 0-5° C. The reaction mixture was stirred atroom temperature for 2 h. The organic layer was separated, washed firstwith water (2×100 mL) and then with saturated sodium carbonate (2×100mL). The organic layer was dried (anhydrous sodium sulfate) and thesolvent removed under reduced pressure at room temperature to obtain theproduct as a colorless oil (89.0 g; Yield: 100%).

ii) Methyl 3-azido-2-hydroxypropionate (16a)

To an ice-cold mixture of methyl 3-amino-2-hydroxypropionatehydrochloride (19) (27.1 g, 0.17 mol) in water (100 mL) anddichloromethane (100 mL) was added sodium carbonate (19.82 g, 0.187 mol)and CuSO₄. 5H₂O (0.3 g, 1.2 mmol) with stirring. Triflylazide fromexperiment 10(B)(i) (crude, 44.6 g, 0.25 mol) was added dropwise keepingthe reaction temperature at 0-5° C. Methanol (about 100 mL) was addeduntil the reaction mixture became homogeneous. The reaction mixture wasallowed to come to room temperature and then stirred for 20 h. Water(500 mL) was added and the aqueous solution thoroughly extracted withdichloromethane (5×100 mL). The combined organic layers were washed withwater (2×200 mL), saturated sodium carbonate (2×200 mL), and then dried(anhydrous sodium sulfate). After removal of the solvent, the residuewas chromatographed over silica gel. Elution with 30% ethyl acetate inhexanes furnished the product as a colorless oil (14.0 g; Yield: 57%).R_(f)0.57 (30% ethyl acetate in hexanes).

C) Methyl 3-azido-2-trifluoromethanesulfonyloxypropionate (16b)

To a solution of methyl 3-azido-2-hydroxypropionate (16a) (14.0 g, 0.097mol) in dry dichloromethane (50 mL) 2,6-lutidine (20.8 g, 0.194 mol) wasadded, followed by triflic anhydride (41.0 g, 0.146 mol) dropwise at 0°C. with stirring under nitrogen over a period of 1 h. The reactionmixture was allowed to come to room temperature and stirred for 16 h.The reaction mixture was diluted with 50 mL of dichloromethane andwashed first with 2N HCl (2×50 mL) and then with water (2×100 mL). Theorganic layer was dried. The solvent was removed under reduced pressureat room temperature and the residue chromatographed over silica gel.Elution with hexane/ethyl acetate (9:1) yielded the product as acolorless oil (20.0 g; yield: 74%). R_(f): 0.7 (30% ethylacetate/hexane).

D) Tris-t-butylN12-(3-azido-2-methoxycarbonyl-1-ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylate

DO3A-tri-t-butyl ester hydrochloride (15a) (1.0 g, 1.81 mmol) wastreated with 1N NaOH (20 mL) and extracted with ether (4×25 mL). Thecombined ether extracts were dried (anhydrous Na₂SO₄), concentratedunder reduced pressure, and dried. DO3A-tris-t-butyl ester (0.8 g, 1.6mmol), thus obtained, was dissolved in dry acetonitrile (5 mL) andanhydrous potassium carbonate (0.28 g, 2 mmol) was added. The mixturewas cooled to 0° C. in an ice-bath. Methyl3-azido-2-trifluoromethanesulfonyloxypropionate (16b) (0.47 g, 1.7mmol), prepared in experiment 3, was added dropwise with stirring undernitrogen. After the addition, the reaction mixture was allowed to cometo room temperature and stirred for 16 h. Acetonitrile was removed underreduced pressure and the resulting residue suspended in 20 mL of waterand extracted with dichloromethane (3×20 mL). The combined organiclayers were washed with water (4×25 mL) and dried (anhydrous sodiumsulfate). The solvent was removed under reduced pressure and theresulting dark red paste was chromatographed over silica gel. Elutionwith 0.5% MeOH in chloroform yielded the product as a pale yellow paste(0.3 g, yield: 29%) Mass Spectrum M+H 642. R_(f): 0.4 (9:1chloroform/MeOH).

E) Tris-t-butylN12-(3-amino-2-methoxycarbonyl-1-ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylate(17a)

To a solution of tris-t-butylN12-(3-azido-2-methoxycarbonyl-1-ethyl)-1,4,7,10-tetracyclododecane-1,4,7-tricarboxylatefrom experiment 10D, (0.6 g, 0.94 mmol) in a 1:1 mixture of t-butanoland methanol (10 mL), palladium on carbon (10%, 0.12 g) was added andthe mixture hydrogenated at 50 p. s. i. until the starting materialdisappeared as per TLC (4 h). The catalyst was filtered off through apad of Celite and the filtrate freed of the solvent. The residue wasdried in vacuo to obtain the product as a colorless paste (0.56 g;yield: 98%). R_(f): 0.12 (9:1 chloroform/MeOH).

EXAMPLE 11N-[1,5-Bis(benzyloxycarbonyl)-3-[2-(benzyloxycarbony)ethyl)]-3-pentyl]-N′-[1,7-bis-(t-butoxycarbony)amino-[4-(3-(t-butyloxycarbonyl)propyl]-4-heptyl]butanedioicdiamide (39)

A) 1,5-Bis(benzyloxycarbonyl)-3-[2-(benzyloxycarbony)ethyl]-3-nitropentane (40)

To a mixture of [3-(2-carboxyethyl)]-3-nitropentane-1,5-dicarboxylicacid (2.8 g, 0.01 mole) (prepared as described by James K. Young et al.,Macromolecules, 1994, 27, 3464-34-71) and cesium carbonate (3.25 g,0.025 mol) in acetonitrile (20.0 mL) was added benzylbromide (8.55 g,6.0 mL, 0.05 mole) and the mixture stirred at RT for 24 h. Inorganicsalts were filtered and the salts washed with acetonitrile. The filtrateand the washings were combined and evaporated to obtain an oil.Purification by chromatography over silica gel (hexane: ethyl acetate,7:3) afforded the benzyl ester (40) as colorless viscous oil (4.5 g,yield: 82%). Mass Spectrum (M+H)hu +=548.

B)3-Amino-[1,5-bis(benzyloxycarbonyl)-3-[2-(benzyloxycarbony)ethyl)]-pentane(41)

1,5-Bis(benzyloxycarbonyl)-3-[2-(benzyloxycarbony)ethyl]-3-nitro pentane(40) (2.9 g, 0.005 mole) was added to aluminum amalgam (prepared from1.0 g of aluminum), in a mixture of THF and water (10:2, 10 mL). Themixture was stirred at room temperature for 6 h. The solvents wereremoved under vacuum and the residue purified by chromatography oversilica gel (hexane: ethyl acetate) to obtain the amine product (41) (2.0g; yield: 77%). Mass Spectrum (M+H)⁺=518. This product was used in thenext step without further purification.

C)N-[1,5-Bis(benzyloxycarbonyl)-3-[2-(benzyloxycarbony)ethyl)]-3-pentyl]-butanedioicmonoamide (42)

To a solution of3-Amino-[1,5-bis(benzyloxycarbonyl)-3-[2-(benzyloxycarbony)ethyl)]-pentane(41) (2.7 g, 0.0049 mole) in pyridine (10.0 mL) was added succinicanhydride (0.5 g, 0.005 mol) and the mixture was stirred at RT for 24 h.Pyridine was removed under vacuum. To the residue water was added andthe solution made acidic with 0.1 N citric acid. The solid formed wasfiltered and air-dried to obtain the monoamide product (2.8 g; yield:85%). Mass Spectrum (M+H)⁺=618. mp. 100-102° C. This product was used inthe next step without further purification.

D)N-[1,5-Bis(benzyloxycarbonyl)-3-[2-(benzyloxycarbony)ethyl)]-3-pentyl]-N′-[1,7-bis-(t-butoxycarbony)amino-[4-(3-(t-butyloxycarbonyl)propyl]-4-heptyl]butanedioicdiamide (39)

To a solution ofN-[1,5-Bis(benzyloxycarbonyl)-3-[2-(benzyloxycarbony)ethyl)]-3-pentylamino-butanedioicmonoamide (42) (0.62 g, 0.01 mol) in dimethylformamide (7 mL) was addedcarbonyldiimidazole (0.165 g, 0.01 mol) and the mixture was stirred atroom temperature for 15 min.1,7-Bis[N-t-butoxycarbonyl)amino]4-[3-(N-t-butoxycarbonyl)-amino)propyl]-4-aminoheptane(0.5 g, 0.001 mol) (prepared as previously described by James K. Younget al., Macromolecules, 1994, 27, 3464-3471) was the added to thereaction mixture and stirred at room temperature overnight.Dimethylformamide was removed in vacuo and the residue treated withwater. The solid that resulted was filtered and air dried.Crystallization from hexane:ethyl acetate furnished the pure diamideproduct (39) (0.52 g; yield: 47%). Mass Spectrum (M+H)⁺=1103. mp 95-96°C.

EXAMPLE 1212-Amino-3,3,9,9-tetramethyl-5-oxa-4,8-diaza-2,10-dodecanedione dioxime(52b)

A) 4-Methyl-1-(phthalimido)-3-pentene (53b)

To a suspension of phthalimide, potassium derivative (11.5 g, 0.062 mol)in dry dimethylformamide (60 mL) was added 1-bromo-4-methyl-3-pentene(10.0 g, 0.061 mol) and the suspension was stirred under N₂ at 90° C.for 4 h. The reaction mixture was poured into water (300 mL) and theprecipitated solid was filtered and washed with water and the solid wasdried under vacuum. Yield: 12.8 g (91%). mp 95-97° C. MS: (M+H)⁺=229.9.

B) 4-Chloro-4-methyl-1-(phthalimido)-3-nitrosopentane (51)

To a cooled (0-5° C.) solution of 4-methyl-1-(phthalimido)-3-pentene(5.0 g, 0.022 mol) and isoamyl nitrite (13.0 g, 15 mL, 0.11 mol) wasadded concentrated hydrochloric acid (4.0 mL, 0.04 mol) with stirring.The reaction mixture was maintained below 5° C. during the addition andstirred at 5° C. for an additional 2 h. The solid formed was filteredand washed with cold ether:ethanol (3:1, 150 mL) and dried. Yield: 4.72g (72.8%). It was crystallized from acetonitrile. mp 140-141° C. MS:(M+H)⁺=296.0.

C) 12-Phthalimido-3,3,9,9-tetramethyl-5-oxa-4,8-diaza-2,10-dodecanedionedioxime (52a)

To a slurry of 3-[[2-(aminoxy)ethyl]amino]-3-methyl-2-butananone oximehydrochloride (50) (2.15 g, 10 mmol) and4-chloro-4-methyl-1-(phthalimido)-3-nitroso-pentane (51) (2.95 g, 10.0mmol) in acetonitrile (100 mL) was added diisopropylethylamine (3.88 g,5.2 mL, 29.8 mmol) and the reaction mixture was stirred at roomtemperature for 24 h. The clear light blue solution obtained aftercompletion of the reaction was concentrated and the residue was treatedwith water. The thick oil obtained was extracted with ether (200 mL),and the ether layer was washed with a saturated solution of sodiumbicarbonate, water and dried (Na₂SO₄). Evaporation of ether gave a foamysolid, which was purified by column chromatography using methylenechloride and methanol (95:5, 90:10). The fractions containing theproduct were collected and evaporated to give a white solid. Yield: 3.1g (71.5%). mp 74-75° C. MS: (M+H)⁺=434.3.

D) 12-Amino-3,3,9,9-tetramethyl-5-oxa-4,8-diaza-2,10-dodecanedionedioxime (52b)

To a solution of12-phthalimido-3,3,9,9-tetramethyl-5-oxa-4,8-diaza-2,10-dodecane dionedioxime (52a) (2.0 g, 4.6 mmol) in methylene chloride (100 mL) was addedhydrazine (0.3 mL, 9.3 mmol) and the reaction mixture was refluxed for 3h. The white solid formed was filtered and the filter cake was washedwith methylene chloride (50 mL). The combined filtrate and the washingswere concentrated to give a thick oil which was dried under vacuum toafford a white solid. Yield: 1.2 g (82%). MS: (M+H)⁺=304.1. This wasused in the next step without further purification.

EXAMPLE 1312-N-(N-Pteroyl-α-L-glutamyl)-3,3,9,9-tetramethyl-5-oxa-4,8-diaza-2,10-dodecanedionedioxime ligand (62)

A)12-N-FMOC-γ-t-Butyl-L-glutamyl-3,3,9,9-tetramethyl-5-oxa-4,8-diaza-2,10-dodecanedionedioxime (58)

To an ice-cooled, stirred solution of commercially available (Bachem)N-FMOC-L-glutamic acid γ-t-butyl ester (57) (0.73 g, 1.65 mmol) indimethylformamide (2.5 mL) was added hydoxybenzotriazole (0.265 g, 1.73mmol) and the solution was stirred for 10 min. Dicyclohexylcarbodiimide(0.357 g, 1.73 mmol) was added and the mixture was stirred at 0° C. for30 min. 12-Amino-3,3,9,9-tetramethyl-5-oxa-4,8-diaza-2,10-dodecanedionedioxime (52b) (0.5 g, 1.65 mmol) was added followed bydiisopropylethylamine (0.23 g, 0.3 mL, 1.73 mmol) and the reactionmixture was stirred at room temperature for 6 h. The precipitateddicyclohexylurea was filtered and the DMF was removed under vacuum. Theresidue was purified by column chromatography on silica gel with CH₂Cl₂:CH₃OH (95:5, 90:10) as the eluant. UV-visible fractions were collectedand solvents were removed to give a viscous oil which was dried undervacuum to give a foamy solid. Yield: 0.45 g (38%). MS: (M+H)⁺=711.5.

B)12-N-L-Glutamyl-γ-t-butyl-3,3,9,9-tetramethyl-5-oxa-4,8-diaza-2,10-dodecanedionedioxime (59)

To a solution of12-N-FMOC-γ-t-butyl-L-glutamyl-3,3,9,9-tetramethyl-5-oxa-4,8-diaza-2,10-dodecanedionedioxime (58) (0.45 g, 0.63 mmol) in acetonitrile (2 mL) was addedpiperidine (0.5 mL) and the mixture was stirred at room temperature for12 h. Acetonitrile was removed on a rotary evaporator and the residuewas purified by silica gel column chromatography using CH₂Cl₂: CH₃OH(95:5, 90:10, 80:20). Fractions containing the product were collectedand the solvent was removed to give a thick oil. It was dried undervacuum to give12-N-L-glutamyl-γ-t-butyl-3,3,9,9-tetramethyl-5-oxa-4,8-diaza-2,10-dodecanediondioxime as a foamy solid. Yield: 0.18 g (58%). MS: (M+H)⁺=489.5.

C)12-N-(N¹⁰-trifluoroacetylpteroyl)-γ-t-butyl-L-glutamyl)-3,3,9,9-tetramethyl-5-oxa-4,8-diaza-2,10-dodecanedionedioxime (60)

To a stirred, 0° C. slurry of N¹⁰-trifluoroacetylpteroic acid (4) (0.125g, 0.3 mmol) in DMF (2.5 mL) was added hydroxybenzotriazole (0.050 g,0.33 mmol). After 10 min, DCC (0.070 g, 0.34 mmol) was added and theslurry was stirred at 0° C. for 15 min. To this suspension was added12-N-L-glutamyl-γ-t-butyl-L-3,3,9,9-tetramethyl-5-oxa-4,8-diaza-2,10-dodecanedionedioxime (59) (0.15 g, 0.3 mmol), followed by diisopropylethylamine (0.13mL, 0.77 mmol). The reaction mixture was allowed to stir for 2 h at 0°C. and then 12 h at RT. DMF was removed under reduced pressure and theresidue was treated with water. The light yellow solid formed wasfiltered and dried under vacuum. The coupled product was purified byreverse phase HPLC (Vydac-C18, 10 μ, 10×25 cm) with a linear gradient of0.1% TFA in H₂O/CH₃CN (0-10% over 10 min and 10-40% over 120 min. Thefractions containing the product were pooled and freeze-dried to give alight yellow solid. Yield: 0.13 g (37%). MS: (M+H)⁺=879.5. HRMS (FAB)m/z, Calcd for C₃₈H₅₄N₁₂O₉F₃: (M+H)⁺:879.4089. Found: 879.4076.

D)12-N-(N-Pteroyl-γ-t-butyl-L-glutamyl)-3,3,9,9-tetramethyl-5-oxa-4,8-diaza-2,10-dodecanedione dioxime (61)

To a solution of12N-(N¹⁰-trifluoroacetylpteroyl)-γ-t-butyl-L-glutamyl)-3,3,9,9-tetramethyl-5-oxa-4,8-diaza-2,10-dodecanedionedioxime (60) (0.31 g, 0.35 mmol) in DMF-Water (4.5:0.5, 5 mL), was addedpiperidine (0.3 mL) and the solution was stirred at RT for 24 h.Dimethylformamide-water were removed under vacuum to give a thick oil,which was treated with water (5 mL). The resulting precipitated yellowsolid was filtered and dried under vacuum. This compound was purified byreverse phase HPLC (Vydac-C18, 10μ, 10×25 cm) with a linear gradient of0.1% TFA in H₂O/CH₃CN (0-10% over 10 min and 10-40% over 120 min. Thefractions containing the product were pooled and freeze dried to give alight yellow solid. Yield: 0.15 g (53%). MS: (M+H)⁺=783.5. HRMS (FAB)m/z, Calcd for C₃₆H₅₄N₁₂O₈ (M+H)⁺:783.4266; Found: 783.4238.

E)12-N-(N-Pteroyl-α-L-glutamyl)-3,3,9,9-tetramethyl-5-oxa-4,8-diaza-2,10-dodecanedionedioxime(62)

12-(N-Pteroyl-γ-t-butyl-L-glutamyl)-3,3,9,9-tetramethyl-5-oxa-4,8-diaza-2,10-dodecanedione dioxime (61) (0.16 g, 0.2 mmol), was dissolved in trifluoroaceticacid (0.3 mL) and stirred for 30 min. Trifluoroacetic acid was removedunder vacuum and the product obtained was purified by reverse phasecolumn chromatography. (Vydac-C18, 10μ, 10×25 cm) with a linear gradientof 0.1% TFA in H₂O/CH₃CN (0-10% over 10 min. and 10-40% over 120 min).The fractions containing the product were pooled and freeze dried togive a light yellow solid. Yield: 0.095 g (48%). MS: (M+H)⁺=727.5. HRMS(FAB) m/z, Calcd for C₃₂H₄₆N₁₂O₈ (M+H)⁺:727.3640; Found: 727.3640.

EXAMPLE 14 99m-Technetium complex of12-(N-Pteroyl-α-L-glutamyl)-3,3,9,9-tetramethyl-5-oxa-4,8-diaza-2,10-dodecanedionedioxime

12-(N-Pteroyl-α-L-glutamyl)-3,3,9,9-tetramethyl-5-oxa-4,8-diaza-2,10-dodecanedionedioxime (2-4 mg) was dissolved in 0.1N NaHCO₃ (0.5 mL) and ^(99m)TcO₄⁻(0.25 mL, 5-15 mCi) was added, followed by 50 μL of a saturatedsolution of stannous tartrate in nitrogen-purged normal saline. After 10minutes, the desired technetium complex was isolated from impurities andexcess ligand by preparative HPLC in ˜45% yield using a YMC basic columnthat was conditioned and eluted with a gradient of MeOH/0.1N trischloride buffer, pH 7.5 at a flow rate of 1.0 mL/min.

EXAMPLE 1512-(N-Pteroyl-γ-L-glutamyl)-3,3,9,9-tetramethyl-5-oxa-4,8-diaza-2,10-dodecanedionedioxime. (Oxa PnAO Folic acid (γ-isomer)) (68)

A)12-N-FMOC-α-t-butyl-L-glutamyl-3,3,9,9-tetramethyl-5-oxa-4,8-diaza-2,10-dodecanedionedioxime. (64)

To an ice cooled stirred solution of N-FMOC-L-glutamic acid-α-t-butylester (63) (0.74 g, 1.74 mmol) and HATU (0.86 g, 2.26 mmol) in methylenechloride (15 mL) was added diisopropylethylamine (0.52 g, 0.75 mL, 4.0mmol). The reaction mixture was stirred under nitrogen at 0° C. for 15min. 12-Amino-3,3,9,9-tetramethyl-5-oxa-4,8-diaza-2,10-dodeanedionedioxime (52b) (0.5 g, 1.65 mmol) was added and the reaction mixture wasstirred at room temperature for 6 h. Methylene chloride was removed andthe residue was treated with water and extracted with methylene chloride(2×50 mL). The methylene chloride solution was washed with sodiumbicarbonate solution (2×50 mL), washed with water and dried (Na₂SO₄).The solvent was removed and the residue was purified by chromatography(silica gel, CH₂Cl₂:CH₃OH, 95:5). Fractions containing the product werecollected and the solvents were removed to give a viscous oil, which wasdried under vacuum to give a foamy solid. Yield: 0.68 g (58%). MS:(M+H)⁺=711.5. HRMS (FAB) m/z, Calcd for C₃₇H₅₄N₆O₈: (M+H)⁺: 711.4081.Found: 711.4109.

B)12-N-L-glutamyl-α-t-Butyl-3,3,9,9-tetramethyl-5-oxa-4,8-diaza-2,10-dodecanedionedioxime (65)

To a solution of12-N-FMOC-α-t-butyl-L-glutamyl-3,3,9,9-tetramethyl-5-oxa-4,8-diaza-2,10-dodecanedionedioxime (64) (0.68 g, 0.96 mmol) in acetonitrile (5 mL) was addedpiperidine (0.5 mL) and the mixture was stirred at RT for 12 h.Acetonitrile was removed on a rotary evaporator and the residue waspurified by silica gel column chromatography using CH₂Cl₂: CH₃OH (95:5,90:10, 85:15). Fractions containing the compound were collected and thesolvent was removed to give thick oil. It was dried under vacuum to give12-N-L-glutamyl-a-t-butyl-3,3,9,9-tetramethyl-5-oxa-4,8-diaza-2,10-dodecanedionedioxime as a foamy solid. Yield: 0.37 g (79%). MS: (M+H)⁺=489.5. HRMS(FAB) m/z, Calcd for C₂₂H₄₄N₆O₆ (M+H)⁺: 489.3401. Found: 489.3376.

C)12N-(N¹⁰-trifluoroacetylpteroyl)-α-t-butyl-L-glutamyl)-3,3,9,9-tetramethyl-5-oxa-4,8-diaza-2,10-dodecanedionedioxime(66)

To a stirred, 0° C. slurry of N¹⁰-trifluoroacetylpteroic acid (4) (0.125g, 0.3 mmol) in DMF (2.5 mL) was added hydroxybenzotriazole (0.050 g,0.33 mmol). After 10 min, DCC (0.070 g, 0.34 mmol) was added and theslurry was stirred at 0° C. for 15 min. To this suspension was added12-N-L-glutamyl-α-t-butyl-L-3,3,9,9-tetramethyl-5-oxa-4,8-diaza-2,10-dodecanedionedioxime (65) (0.15 g, 0.3 mmol) followed by diisopropylethylamine (130.0mL, 0.77 mmol). The reaction mixture was allowed to stir for 2 h at 0°C. and then 12 h at room temperature. DMF was removed under reducedpressure and the residue was treated with water. The light yellow solidformed was filtered and dried under vacuum. The coupled product waspurified by reverse phase HPLC (Vydac-C18, 10μ, 10×25 cm) with a lineargradient of 0.1% TFA in H₂O/CH₃CN (0-10% over 10 min and 10-40% over 120min. The fractions containing the product were pooled and freeze-driedto give a light yellow solid. Yield: 0.16 g (59%). MS: (M+H)⁺=879.5.HRMS (FAB) m/z, Calcd for C₃₈H₅₄N₁₂O₉F₃ (M+H)⁺: 879.4089; Found:879.4091.

D)12-(N-Pteroyl-α-t-butyl-L-glutamyl)-3,3,9,9-tetramethyl-5-oxa-4,8-diaza-2,10-dodecanedione dioxime (67)

To a solution of12N-(N¹⁰-trifluoroacetylpteroyl)-α-t-butyl-L-glutamyl)-3,3,9,9-tetramethyl-5-oxa-4,8-diaza-2,10-dodecanedionedioxime (66) (0.25 g, 0.28 mmol) in DMF-Water (4.5:0.5 mL, 3 mL) wasadded piperidine (0.25 mL) and the solution was stirred at roomtemperature for 24 h. Dimethylformamide-water was removed under vacuumto give a thick oil. The oil was treated with water (5 mL) and theprecipitated yellow solid was filtered and dried under vacuum. Yield:0.2 g (90%). An analytical sample was purified by reverse phase HPLC(Vydac-C18, 10μ, 10×25 cm) with a linear gradient of 0.1% TFA inH₂O/CH₃CN (0-10% over 10 min and 10-40% over 120 min). The fractionscontaining the product were pooled and freeze-dried to give a lightyellow solid. MS: (M+H)⁺=783.5. HRMS (FAB) m/z, Calcd for C₃₆H₅₄N₁₂O₈(M+H)⁺: 783.4266; Found: 783.4240.

E)12-(N-Pteroyl-γ-L-glutamyl)-3,3,9,9-tetramethyl-5-oxa-4,8-diaza-2,10-dodecanedionedioxime (68)

12-(N-Pteroyl-α-t-butyl-L-glutamyl)-3,3,9,9-tetramethyl-5-oxa-4,8-diaza-2,10-dodecanedionedioxime (67) (0.135 g) was dissolved in trifluoroacetic acid (0.3 mL)and stirred for 30 min. Trifluoroacetic acid was removed under vacuumand the product obtained was purified by reverse phase columnchromatography. (Vydac-C18, 10μ, 10×25 cm) with a linear gradient of0.1% TFA in H₂O/CH₃CN (0-10% over 10 min and 10-30% over 90 min). Thefractions containing the product were pooled and freeze-dried to give alight yellow solid. Yield: 0.065 g (48%). MS: (M+H)⁺=727.5. HRMS (FAB)m/z, Calcd for C₃₂H₄₆N₁₂O₈ (M+H)⁺: 727.3640; Found: 727.3659.

EXAMPLE 16 99m-Technetium Complex of12-(N-Pteroyl-γ-L-glutamyl)-3,3,9,9-tetramethyl-5-oxa-4,8-diaza-2,10-dodecanedionedioxime

12-(N-Pteroyl-γ-L-glutamyl)-3,3,9,9-tetramethyl-5-oxa-4,8-diaza-2,10-dodecanedionedioxime (2-4 mg) was dissolved in 0.1N NaHCO₃ (0.5 mL) and ^(99m)TcO₄⁻(0.25 mL, 5-15 mCi) was added, followed by 50 μL of a saturatedsolution of stannous tartrate in nitrogen-purged normal saline. After 10minutes at room temperature, the desired technetium complex was purifiedfrom impurities and excess ligand by preparative HPLC using a YMC basiccolumn that was conditioned and eluted with a gradient of MeOH/0.1N trischloride buffer, pH 7.5 at a flow rate of 1.0 mL. The desired productwas isolated in ˜45% yield.

B. Biological Evaluation

EXAMPLE 17 Binding Studies with ¹⁵³Gd-DO3A-APA-folate (α or γ isomer) inKB and JAR Cells in Vitro

Cell culture: KB cells (a human nasopharyngeal epidermal carcinoma cellline) and JAR cells (a human choriocarcinoma cell line) were obtainedfrom ATCC (American Type Culture Collection). Both cell lines arereported to overexpress transcripts encoding folate binding protein. KBcells were grown in folate-free Minimal Essential Medium with Earle'ssalts, 1-glutamine and non-essential amino acids obtained from LifeTechnologies, Inc. JAR cells were grown in RPMI formulated without folicacid, (catalog #27016, Life Technologies, Inc). The media for each cellline was supplemented with 10% defined fetal calf serum (HyClone, Inc.)Monolayers were cultured at 37° C. in a humidified atmosphere containing5% CO₂.

Forty-eight hours prior to each experiment, 5×10⁵ cells were seeded into35 mm culture dishes and allowed to grow for 2 days in folate-depletedmedia. The cells were then washed with Tris-buffered saline (TBS=150 mMNaCl, 50 mM Tris, pH 7.5) and 1 mL of fresh media containing 10 pmol¹⁵³Gd-DO3A-APA-folate (α or γ isomer) and 0-900 pmol of cold nativefolate per sample was added. Control experiments were performed using 10pmol of ³H folate (Amersham International) and 0-900 pmol of cold nativefolate. The cells were incubated for 30 min at 37° C. They were thenwashed three times with ice cold TBS and suspended in one ml of water.Cell associated radioactivity was determined by gamma (¹⁵³Gd-folate) orscintillation counting (³H-folate) of a 500 μl aliquot of thissuspension; 100 μl was used in the BCA protein assay (Pierce catalog#23225), which was used to determine and normalize for cellular proteincontent.

Data from these studies in KB cells are shown in FIG. 1. In KB cells,the data demonstrate that ³H folate and the complexes of this inventionwere equally effective in their ability to compete with cold nativefolate for binding to KB cells that overexpress folate binding protein.No significant differences were seen in the amount of ³H-folate or¹⁵³Gd-DO3A-APA- (α)- or (γ)-folate isomers that were bound to the KBcells. These results indicate that covalent attachment of a metalchelate to either the alpha or gamma carboxylate of folic acid does notcompromise the ability of the conjugate to bind to KB cells. The resultsobtained for the alpha isomer are surprising in light of Wang et al.,who, as noted above, have taught that alpha conjugates show no abilityto compete with native folate.

In a second experiment, the ability of the alpha and gamma isomers of¹⁵³Gd-DO3A-APA-folate to bind to KB cells at 4° C. was studied. KB cells(˜5×10⁵ cells/well) were seeded into 35-mm wells and incubated for 48hours as described above. The cells were then cooled to 4° C. for 30 minand incubated with the following mixtures for 30 min at 4° C.:

-   -   1. 10 pmol of ³H-folate    -   2. 10 pmol of ³H-folate and 250 pmol of native folate    -   3. 10 pmol of ¹⁵³Gd/Gd(DO3A-APA-α-folate)    -   4. 10 pmol of ¹⁵³Gd/Gd(DO3A-APA-α-folate) and 250 pmol of native        folate    -   5. 10 pmol of ¹⁵³Gd/Gd(DO3A-APA-γ-folate)    -   6. 10 pmol of ¹⁵³Gd/Gd(DO3A-APA-γ-folate) and 250 pmol of native        folate

Following the incubation period, the cells were washed 3 times withice-cold Tris-buffered saline. The cells were then stripped from theplates using 1.0 mL of water. Aliquots of the water/cell mixture wereassayed for the respective radioisotopes and for cellular protein (todetermine the number of KB cells present in each well). The radioassaydata were used to calculate the % of radiolabeled compound bound to thecells in the absence and presence of unlabeled native folate. Theresults from this study are given in FIG. 2. Data in FIG. 2 arepresented as the percentage bound, relative to the % bound in thecontrol wells containing 10 pmol of ³H-folate.

These data indicate that when 10 pmol of ¹⁵³Gd/Gd(DO3A-APA-folate) wasincubated with KB cells for 30 min at 4° C., the α- and γ-isomers of¹⁵³Gd/Gd(DO3A-APA-folate) both bound to the KB cells to the same extentas that observed with 10 pmol of ³H-folate. (FIG. 2, dark bars).

These binding experiments were repeated in the presence of 250 pmol ofcold native folate, to determine if the addition of excess cold folatewould cause a decrease in the quantity of ³H or ¹⁵³Gd bound to the KBcells at the end of the experiment. Such a result is expected if theradiolabeled compounds and native folate compete for binding to folatebinding protein on the KB cells and an excess of cold folate is added.The results observed (FIG. 2, white bars) indicate that both nativefolate and the alpha and gamma isomers of ¹⁵³Gd/Gd(DO3A-APA-folate) docompete for folate binding protein on the KB cells, and that theaddition of 250 pmol of cold folate causes a similar effect on thedegree of binding of 10 pmol ³H-folate as it does on the binding of 10pmol of either the alpha or gamma isomer of ¹⁵³Gd/Gd(DO3A-APA-folate).

Data from studies in JAR cells are shown in FIG. 3.

In these cells, the (α)- or (γ)-isomers of ¹⁵³Gd-DO3A-APA-folate wereequally effective in their ability to compete with cold native folatefor binding to the JAR cells. In contrast to the data from KB cells,binding of these Gd-folate compounds to JAR cells was about half aseffective as that of the radiolabeled control (³H-folate) undercomparable test conditions, as shown in FIG. 1A.

The finding that the alpha and gamma isomers are equivalent in theirability to compete with native folate for binding to the JAR cells isagain surprising, as the results are contrary to the recent patentpublication of Low, Green, et al. (WO 96/36367) which teaches away fromthe alpha isomer being a compound that is taken up by folate uptakemechanism(s) such that it could be used to image the distribution of thesame. Even more surprising was the result obtained with theBis(DO3A-APA)folate compound, which contains a metal chelate moiety atboth the alpha and gamma carboxylates of folate; at some folateconcentrations, binding of this compound was higher than that observedfor the ³H-folate control.

EXAMPLE 18 In Vitro Efflux Studies with ³H folate, ¹⁵³Gd-DO3A-APA-(α)-and -(γ)-folate

Efflux studies were performed to examine the washout of Gd-folates ornative folate from KB and JAR cells in vitro. Briefly, 250 pmol of³H-folate, ¹⁵³Gd-DO3A-APA-(γ)-folate or ¹⁵³Gd-DO3A-APA-(α)-folate wasincubated with approximately 100,000 KB or JAR cells for 30 minutes at37° C., 5% CO₂. Under these conditions, KB and JAR cells were previouslyshown to reach saturation for folate uptake/association. At the end ofthe 30-min incubation period, the cells were washed five times withice-cold media lacking folate.

These folate-loaded cells were then incubated at 37° C. for 24 hourswith folate-depleted media. At predetermined intervals during this time,the incubation medium was removed and replaced with fresh medium at 37°C. The radioactivity present in each aliquot of the incubation mediumwas used to determine the rate at which radioactivity from the³H-folate, ¹⁵³Gd-DO3A-APA-(γ)-folate or ¹⁵³Gd-DO3A-APA-(α)-folate washedout of the cells. At the end of the study, the cells were suspended inone ml of water and assayed for radioactivity and protein content.

The results of washout studies with the alpha and gamma isomers ofGd(DO3A-APA)-folate and native folate are compared in FIGS. 3 and 3A.The data show that when there is no added folate in the media, washoutfrom KB and JAR cells is faster for the alpha derivative than it is forthe gamma isomer or native substrate. This finding may be relevant tothe in vivo situation where the levels of extracellular folate will dropafter the initial bolus.

EXAMPLE 19 In Vitro Exchange Studies with ³H-folate, ¹⁵³Gd-DO3A-APA-(α)-and -(γ)-folate

Exchange studies were performed to examine the effect of native folateon the radioactivity levels in KB or JAR cells that had been pre-loadedwith a saturating level of radiolabeled Gd-folates or native folate invitro. ³H-folate, ¹⁵³Gd-DO3A-APA-(γ)-folate or ¹⁵³Gd-DO3A-APA-(α)-folate(250 pmol) was incubated with KB or JAR cells as described in Example18. At the end of the 30-min incubation period, these ¹⁵³Gd-folate or³H-folate-loaded cells were washed five times with ice-cold mediacontaining 250 nM cold, native folate and were then incubated at 37° C.for 24 hours with media that contained 250 pmol of cold folate. Atpredetermined intervals during this time, the incubation medium wasremoved and replaced with fresh medium at 37° C. The radioactivitypresent in each aliquot of the incubation medium was used to determinethe rate at which radioactivity from the ³H-folate,¹⁵³Gd-DO3A-APA-(γ)-folate or ¹⁵³Gd-DO3A-APA-(α)-folate washed out of thecells. At the end of the study, the cells were suspended in one ml ofwater and assayed for radioactivity and protein content.

The results of these exchange studies in KB cells are shown in FIG. 4A.KB cells that were loaded with a saturating dose of¹⁵³Gd-DO3A-APA-(α)-folate and subsequently incubated with solutionscontaining cold native folate lost close to 100% of their radioactivityto the incubation bath within 24 hours, suggesting that the alpha isomerexchanges with the native folate present in the external medium. Incontrast, KB cells loaded with ³H folate or Gd-DO3A-APA-(γ)-folate lostonly 20% of the initial radioactivity in the cells to the medium over 24hours.

The results of the exchange studies in JAR cells are shown in FIG. 4B.In this cell line, the alpha compound again showed a faster initial netrate of loss of radiolabel to the media than was observed with the gammaand native folate compounds. After 6 hours, 60% of the radioactivityfrom the cells loaded with the alpha isomer was found in the medium,whereas only 30% of the starting activity had been lost from JAR cellsthat were loaded with either Gd(DO3A-APA)-folate gamma isomer or ³Hfolate. After 24 hours the gamma isomer and native folate also showedsignificant net loss of activity from the JAR cells to the medium. At 24hours the amounts of alpha and gamma compound that had washed from thecell were similar.

These studies demonstrate that the clearance properties of the compoundderivatized at the alpha carboxylate of folate are significantlydifferent from those of the gamma analog or underivatized native folate.In vivo, this property may be advantageous as it may serve to reduce thehalf-life of radioactivity in cells that bind folate analogs, which mayimprove the dosimetry properties of the agent. For applications in MRimaging this property may be advantageous as it may serve to reduce thebiological half-life of the agent in cells that bind folate analogs,which may improve the toxicological profile of the agent.

EXAMPLE 20 Biodistribution Studies in Tumor-bearing Nude Mice at“Tracer” Doses of Gd-DO3A-APA-(α)-folate, Gd-DO3A-APA-(γ)-folate andBis(Gd-DO3A-APA)-folate

To define the ability of the complexes of this invention to targetfolate-binding protein-expressing tumor cells in vivo, distributionstudies were conducted in female athymic (Nu/Nu) mice that wereimplanted with KB or JAR tumors. The mice were maintained on an adlibitum folate-depleted diet (gamma-irradiated Purina folate-deficientbasal diet 5831C-2 with 1% succincyl sulphathiazole) beginning two weeksprior to inoculation. This diet was maintained throughout the durationof the study. The mice were inoculated subcutaneously in the scapularregion with 0.1 mL of a tumor cell suspension containing 4×10⁶ KB or JARcells. When tumors had grown to 0.2-0.4 cm³ (2-4 weeks), animals wereanesthetized (100 mg ketamine/kg, i.m. and 10 mg xylazine/kg i.p.) and a“trace” dose (0.08 nmol/kg) of either ¹⁵³Gd-DO3A-APA-(α)-folate,¹⁵³Gd-DO3A-APA-(γ)-folate or Bis(¹⁵³Gd-DO3A-APA)-folate in a volume of0.2 mL was delivered via tail-vein injection (n=3 animals/compound).Thirty minutes post injection, the mice were sacrificed and selectedorgans were removed, weighed, and assayed for radioactivity, in order todetermine the % injected dose/g-tissue (% ID/g) and the % injecteddose/organ (% ID/organ), following procedures known to those skilled inthe art. The 30 min tumor and kidney biodistribution results obtainedfollowing a tracer (0.08 nmol/kg) dose of Gd-folate are shown in TableI. These data show that the % ID/g in tumor is comparable for all threecompounds studied. This result is surprising in light of Low, Green etal, who teach that folates derivatized in the alpha position have littleaffinity for KB cells in vitro, and by extension, in vivo. The datafurther show that the % ID/g in kidney is higher forGd-DO3A-APA-(γ)-folate than it is for the two compounds that arederivatized in the alpha position (Gd-DO3A-APA-(α)-folate andBis(Gd-DO3A-APA)-folate.

TABLE I Distribution data: KB Mice Gd-DO3A- Bis(Gd-DO3A- Gd-DO3A- (N =3/data set @ 30 min) APA- APA)- APA- (0.08 nmol/kg) (α)-folate folate(γ)-folate % ID/g-tumor 7.7 (1.3) 5.9 (0.8) 8.9 (2.0) % ID/g kidneys39.0 (2.4) 47 (6) 58.4 (8.3)

These results suggest that the radiation dose to kidney that is providedby the two radiolabeled folate compounds that are derivatized in thealpha position will be lower than that provided by the compound that isonly derivatized at the gamma carboxylate. Improved kidney dosimetry isattained without adversely affecting tumor radioactivity levels, whichare roughly comparable for all three compounds. As radiation backgroundfrom kidney will interfere with the ability to image folate-receptorpositive tissues in adjacent tissues (e.g. ovarian and uterine tumors),substitution at the alpha position may provide a distinct advantage.Implications for the radiation dose provided by alpha-substitutedradiotherapeutic folate derivatives to target and non-target tissues areobvious.

Improved results with alpha-derivatized complexes were also seen in astudy that compared the levels of radioactivity found in the kidney andurine following a 0.08 nmol/kg dose of one of the three radiolabeled¹⁵³Gd-folate derivatives. Data obtained at 30 and 60 minutes postinjection are shown in Table II.

TABLE II Compound: % ID/Organ (S.D.) at 30 or 60 min Post Injection doseKidneys: Urine/Bladder: 0.08 nmol/kg 30 min 60 min 30 min 60 minGd-DO3A-APA- 116.5 (1.1) 21.0 (1.8) 23.3 (5.2) 21.6 (2.4) (α)-folate (N= 3,KB) (n = 2,JAR) (N = 3,KB) (N = 2,JAR) Bis(Gd-DO3A- 17.5 (1.7) 17.8(3.0) 18.5 (7.7) 30.3 (5.0) APA)-folate (N = 6,KB/JAR) (N = 2,JAR) (N =6,KB/JAR) (N = 2,JAR) Gd-DO3A-APA- 19.8 (1.1) 34.3 (7.0) 3.3 (9.6) 5.2(1.6) (γ)-folate (N = 3,KB) (N = 3,JAR) (N = 3,KB) (N = 2,JAR)

Radioactivity in the kidney following injection of the gamma-folatederivative was higher than that for the alpha and bis derivatives atboth 30 and 60 minutes. The amount of radioactivity in the urine andbladder for the alpha compounds was different from that observed withthe gamma isomer. Between 20 and 30% of the radioactivity was excretedfor the alpha and bis compounds over one hour. In contrast thegamma-Gd(folate) compound did not show any appreciable renal excretioneven at 60 min.

EXAMPLE 21 Biodistribution Studies in Tumor-bearing Nude Mice at “MRI”Dose Levels (0.1 mmol/kg) of ¹⁵³Gd/Gd-DO3A-APA-(α)-folate,¹⁵³Gd/Gd-DO3A-APA-(γ)-folate and Bis(¹⁵³Gd/Gd-DO3A-APA)folate

Biodistribution studies were performed using female nu/nu mice implantedwith subcutaneous JAR tumors using the general procedures described inthe example above. Animals were sacrificed 60 min post IV injection ofan “MRI” dose level (0.1 mmol/kg) of ¹⁵³Gd/Gd-DO3A-APA-(α)-folate,¹⁵³Gd/Gd-DO3A-APA-(γ)-folate or Bis(¹⁵³Gd/Gd-DO3A-APA)folate andselected organs were removed, weighed, and assayed for radioactivity, inorder to determine the % injected dose/g-tissue (% ID/g) in the tumors.The data obtained in JAR tumors are shown in Table III.

TABLE III [Gd] in Mean % ID/g-JAR Tumor Tumor tumor tissue (60 min) 100μmol/kg dose Tissue % ID/g (μM) ¹⁵³Gd/Gd-DO3A-APA-(α)-folate JAR 2.43(0.71) 55 (20) (N = 4) Tumor ¹⁵³Gd/Gd-DO3A-APA-(γ)-folate JAR 1.83(0.87) 44 (20.8) (N = 3) Tumor Bis(¹⁵³Gd/Gd-DO3A-APA)folate JAR 1.48(0.12) 55 (4) (n = 3) (79.2 μmol/kg dose) Tumor

The % ID/g values in tumor 60 min post injection of a 0.1 mmol/kg dosewere comparable for the Gd-DO3A-APA-(α) and (γ)-folate and Biscomplexes. This result was surprising in light of the reports of Wang etal. and the patent of Low et al. as uptake of the alpha derivatizedcompounds would not be expected based on literature teachings. Theconcentration of gadolinium in these tumors was calculated from theobserved % ID/g values, and it was found the gadolinium concentrationsachieved in the tumors with all three Gd-folate compounds weresufficient to provide detectable enhancement of the MRI signal from thetumor. Kidney localization was also noted for all three compounds,suggesting MRI applications for these compounds in selective enhancementof the signal intensity from kidney as well as tumor.

EXAMPLE 22 Biological Evaluation of the Alpha and Gamma Isomers of^(99m)Tc-oxa-folate in KB Cells

The ability of the alpha and gamma isomers of ^(99m)Tc-oxa-folate tobind to KB cells was studied at 4° C. KB cells (˜5×10⁵ cells/well) wereseeded into 35-mm wells and incubated for 48 hours as described inexample 17 above. The alpha and gamma isomers of ^(99m)Tc-oxa-folate,were prepared and purified as described in examples 14 and 16. The cellswere then cooled to 4° C. for 30 min and incubated with the followingmixtures for 30min at 4° C.:

-   -   1. 10 pmol of ³H-folate    -   2. 10 pmol of ³H-folate and 250 pmol of native folate    -   3. Trace amounts (˜0.5 μCi) of ^(99m)Tc-Oxa-α-folate    -   4. Trace amounts (˜0.5 μCi) of ^(99m)Tc-Oxa-α-folate and 260        pmol of native folate    -   5. Trace amounts (˜0.5 μCi) of ^(99m)Tc-Oxa-γ-folate    -   6. Trace amounts (˜0.5 μCi) of ^(99m)Tc-Oxa-γ-folate and 260        pmol of native folate

Following the incubation period, the cells were washed 3 times withice-cold Tris-buffered saline. The cells were then stripped from theplates using 1.0 mL of water. Aliquots of the water/cell mixture wereassayed for the respective radioisotopes and for cellular protein (todetermine the number of KB cells present in each well). The radioassaydata were used to calculate the % of radiolabeled compound bound to thecells in the absence and presence of unlabeled native folate. Theresults from this study are given in FIG. 4. Data in FIG. 4 arepresented as the percentage bound, relative to the % bound in thecontrol wells containing 10 pmol of ³H-folate.

These data indicate that when ^(99m)Tc-Oxa-folate was incubated with KBcells for 30 min at 4° C., the γ-isomer of ^(99m)Tc-Oxa-folate bound tothe KB cells to the same extent as that observed with 10 pmol of³H-folate; binding observed with the alpha isomer of ^(99m)Tc-Oxa-folatewas 140% of that observed with 10 pmol of ³H-folate. (FIG. 4, darkbars).

These binding experiments were repeated in the presence of 250-260 pmolof cold native folate, to determine if the addition of excess coldfolate would cause a decrease in the quantity of ³H or ¹⁵³Gd bound tothe KB cells at the end of the experiment. Such a result is expected ifthe radiolabeled compounds and native folate compete for binding tofolate binding protein on the KB cells and an excess of cold folate isadded. The results observed (FIG. 4, white bars) indicate that bothnative folate and the alpha and gamma isomers of ^(99m)Tc-Oxa-folate docompete for folate binding protein on the KB cells, and that theaddition of 250 pmol of cold folate causes a similar effect on thedegree of binding of 10 pmol ³H-folate as it does on the binding of 10pmol of either the alpha or gamma isomer of ^(99m)Tc-Oxa-folate.

Having described the invention, it is understood that changes andmodifications may be effected within the spirit and scope of theinvention.

1. A diagnostic, therapeutic or radiotherapeutic or chemotherapeuticcomposition for visualization, therapy, chemotherapy or radiotherapy oftissues or organs that overexpress folate-binding protein comprising: a)folate-receptor binding ligand comprising one or more folate-receptorbinding moieties, at least one of which is conjugated through its alphacarboxylate via an optional linking group to one or more macrocyclic ornon-macrocyclic metal-chelating ligand radicals that are optionallychelated to paramagnetic, superparamagnetic, radioactive ornon-radioactive metals for detection outside the body by imaging meansfor diagnosis or for providing a therapeutic, chemotherapeutic, orradiotherapeutic effect; wherein said folate receptor binding ligand hasthe structure of formula II:

 wherein R₀ is a folate-receptor binding residue of formula:

each X is independently —0—, —S—, —NH—, or —NR₁—: n1 is 0 or 1; b1 is 1to 3; m1 is 1 to 81; each K₁ is independently a) a macrocyclic ornon-macrocyclic metal-chelating ligand radical that is optionallychelated to a paramagnetic, superparamagnetic, radioactive ornon-radioactive metal M₁, or b) a chemotherapeutic drug; —K₂ is —H,-alkyl, -alkenyl, -alkynyl, -alkoxy, -aryl, -alkyl, —CON(R₂)₂,-glutamate, -polyglutamate, or —K₃; —K₃ is

wherein —K₅ is either a) a macrocyclic or non-macrocyclicmetal-chelating ligand that is optionally chelated to a paramagnetic,superparamagnetic, radioactive or non-radioactive metal M₅, or b) achemotherapeutic drug n5 is 0 or 1; b5 is 1 to 3; m5 is 1 to 81; —(A)p—and —(A)p*— are each independently optional linkers comprising astraight or branched chain wherein the moieties “A” are the same ordifferent and selected from the group consisting of: —CH₂—, —CHR₃—,—CR₄R₅—, —CH═CH—, —CH═CR₆—, >CR₇—CR₈<, —C═C—, —CR₉═CR₁₀—, —C≡C—,-cycloalkylidene-, -cycloalkenyl-, -arylidene-, -heterocyclo-, carbonyl(—CO—), —O—, —S—, —NH—, —HC═N—, —CR₁₁═N—, —NR₁₂—, —CS—,

 and p and p* are independently 0 to 24, or —X—[(A)]p— and —X—[(A)p]*-may each independently be the group —Q— wherein —Q— is—[C(R′)(R″)]_(s1)—[C(t)(R₂₁)]_(s2)—[C(R₂₂)(R₂₃)]_(s3)—X3-Y—X4—; whereineach s1, s2, s3, and s4 is independently 0 to 2; each X3, X4, X5, and X6is independently a single bond, —O—, —S—, or —N(R₂₄)—; Y is a singlebond, —C(R₂₅)(R₂₆)—, or Y1 wherein, Y1 is —C(═X5)—X6—W—, wherein W is asingle bond, -alkylidene-, -cycloalkylidene-, -arylidene-,alkenylidene-, or -alkynylidene-, whose carbon atoms may or may not besubstituted; t is H, R₂₇, —C(O)OR₂₈, —P(O)(OR₂₉))OH, —P(O)(OR₃₀))OR₃₁,—P(O)(OR₃₂)R₃₃, —P(O)(OH)R₃₄, —C(O)N(R₃₅)(R₃₆), or C(O)NH(R₃₇); each R′and R″ is independently a single bond, H, alkyl, alkoxy, cycloalkyl,hydroxyalkyl, aryl, or heterocyclo, each of which is optionallysubstituted, each R₃ through R₅, R₇, R₈, R₂₁ through R₂₃, and R₂₅through R₂₇ is independently H, alkyl, alkoxy, halogen, hydroxy,cycloalkyl, hydroxyalkyl, aryl, or heterocyclo, each of which isoptionally substituted; each R₁, R₂, R₆, R₉ through R₁₂, R₂₄, and R₂₈through R₃₇ is independently H, alkyl, alkenyl, cycloalkyl, aryl, a 5-or 6- membered nitrogen or oxygen containing heterocycle; wherein —K₂ is

and both —K₁ and —K₅ are macrocyclic or non-macrocyclic metal chelatesthat are each optionally chelated to radioactive, nonradioactive,paramagnetic or superparamagnetic metals M₁ or M₅; wherein —[(A)p]—K₁and —[(A)p*]—K₅ are each in their entirety, polydentate ligands radicalsof formula IIIa-IIIc;

 wherein Q is the group—(C(RR))_(m1)—Y¹(C(RR))_(m2)—(Y²—(C(RR))_(m3))_(n−), wherein Y¹ and Y²are independently —CH₂—, —NR—, —O—, —S—, —SO—, —SO₂— or —Se—; n is 0 or1; and m1, m2 and m3 are integers independently selected from 0 to 4,provided that the sum of m1 and m2 is greater than zero; all R and R*groups are independently —R₄, —Cl, —F, —Br, —OR⁵, —COOR⁵, —CON(R⁵)₂,—N(R⁵)₂, -alkyl-COOR⁵, -alkyl-C(O)—N(R⁵)₂; -alkyl-N(R⁵)₂; —C(O)OR⁵;—C(O)N(R⁵)₂; -aryl-N(R⁵)₂; acyl; acyloxy; heterocyclo; hydroxyalkyl;—SO₂—R⁵; -alkyl-SO₂—R⁵; or —R³; wherein each —[R³]— is, in its entirety,the linking group —[(A)p]— or —[(A)p*]— that serves to couple the metalchelating ligand radical to —X—; each —R⁴ is independently —H, -alkyl,-alkoxy, -hydroxy, -cycloalkyl, hydroxyalkyl, -aryl, or -heterocyclo,each of which is optionally substituted; each —R⁵ is independently —H,-alkyl, -aryl, -cycloalkyl or -hydroxyalkyl, each of which isindependently substituted; with the provisos that a carbon atom bearingan R group is not directly bonded to more than one heteroatom; and atleast one R or R* group on each —K₁ and —K₅ is —[R³]—; or apharmaceutically acceptable salt thereof; in a pharmaceuticallyacceptable carrier.
 2. The composition of claim 1 wherein both —K₁ and—K₅ are metal-chelating ligand radicals of formula V:

wherein —Q— is the group—(C(RR))_(m1)—(Y¹)_(n)—(C(RR))_(m2)—(Y²—(C(RR))_(m3))_(n1); Y¹ and Y²are each independently —CH₂—, —NR⁵—, —O—, —S—, —SO—, —SO₂— or —Se—; nand n1 are each independently 0 or 1; and m1, m2 and m3 areindependently 0 or an integer from 1 to 4; provided that m1 and m2 arenot both 0, that m1+m2+n+n1 is less than 6 and that a carbon atombearing an R group is not directly bonded to more than one heteroatom;each —R and —R* group is independently: —R⁴; -alkoxy; -hydroxy;-halogen, especially fluoro; -haloalkyl; —OR⁵; —C(O)—R⁵, —C(O)—N(R⁵)₂,—N(R⁵)₂, —N(R⁵)—COR⁵; -alkyl-C(O)—OR⁵, -alkyl-C(O)—N(R⁵)₂,-alkyl-N(R⁵)₂—, -alkyl-N(R⁵)—COR⁵, -aryl-C(O)—OR⁵, -aryl-C(O)—N(R⁵)₂,aryl-N(R⁵)₂—, -aryl-N(R⁵)—COR⁵, -nitrile, -acyl, -acyloxy, -heterocyclo,-hydroxyalkyl, alkoxyalkyl, hydroxyaryl, arylalkyl, —SO₂—R⁵,-alkyl-SO₂—R⁵, or —[R³]— wherein each —[R³]— is, in its entirety, thelinking group —[(A)p]— or —[(A)p*]— that serves to couple the metalchelating ligand radical —K₁ or —K₅ to —X—; each —R⁴ is independently—H, -alkyl, -alkoxy, -hydroxy, -cycloalkyl, -hydroxyalkyl, -aryl, or-heterocyclo, each of which is optionally substituted; each —R⁵ isindependently —H, -alkyl, -aryl, -cycloalkyl or -hydroxyalkyl, each ofwhich is optionally substituted; or two R groups, or an R group and anR* group, taken together with the one or more atoms to which they arebonded, form a saturated or unsaturated, spiro or fused, carbocyclic(such as fused 1,2-phenyl) or heterocyclic ring which may beunsubstituted or substituted by one or more groups R or R* groups above;each G¹ and G² is independently —OH or —(NR⁶)₂; with the proviso that atleast one of G¹ or G² is —(NR⁶)₂, where each R⁶ is independentlyhydrogen, alkyl, aryl, acyl or —[R³]—; and A is a linking group; and pis 0 or a positive integer; with the proviso that at least one R , R*,or R⁶ group is —[R³]—; or a pharmaceutically acceptable salt thereof. 3.A diagnostic, therapeutic or radiotherapeutic composition forvisualization, therapy or radiotherapy of tissues or organs thatoverexpress folate-binding protein using nuclear medicine, magneticresonance imaging or neutron capture radiotherapy applicationscomprising; a) a folate-receptor binding ligand and b) apharmaceutically acceptable carrier wherein said folate-receptor bindingligand has the structure of formula IIb:

 wherein —K₁ is —H, -alkyl, -alkenyl, -alkynyl, -alkoxy, -aryl, -alkyl,—CON(R₂)₂, -glutamate, or -polyglutamate; —K₅ is a polydentate metalchelating ligand; M₅ is a radioactive, paramagnetic or superparamagneticmetal; each —X— is independently —O—, —S—, —NH—, or —NR₁—; b5=1 to 3,m5=1; n5 is 0 or 1; —R₀ is a folate-receptor binding residue of formula:

each —[(A)p*]— is an optional linker independently comprising a straightor branched chain made up of “p*” individual (A) moieties that are thesame or different and are selected from the group consisting of: —CH₂—,—CHR₃—, —CR₄R₅—, —CH═CH—, —CH═CR₆—, >CR₇—CR₈<, >C═C<, —CR₉═CR₁₀—, —C≡C—,-cycloalkylidene-, -cycloalkenyl-, -arylidene-, -heterocyclo-, carbonyl(—CO—), —O—, —S—, —NH—, —HC═N—, —CR₁₁═N—, —NR₁₂—, —CS—, and

 and p* is 0 to 24; or —X—[(A)]p*— is, in its entirety, the group—Q—wherein —Q— is—[C(R′)(R″)]_(s1)—[C(t)(R₂₁)]_(s2)—[C(R₂₂)(R₂₃)]_(s3)—X3—Y—X4—; whereins1, s2, s3, and s4 are independently 0 to 2; X3, X4, X5, and X6 areindependently a single bond, —O—, —S—, or —N(R₂₄)—; Y is a single bond,—C(R₂₅)(R₂₆)—, or —Y1— wherein Y1 is —C(═X5)—X6—W—, wherein W is asingle bond, -alkylidene-, -cycloalkylidene-, -arylidene-,-alkenylidene-, or -alkynylidene-, whose carbon atoms are optionallysubstituted; t is H, R₂₇, —C(O)OR₂₈, —P(O)(OR₂₉))OH, —P(O)(OR₃₀))OR₃₁,—P(O)(OR₃₂)R₃₃, —P(O)(OH)R₃₄—C(O)N(R₃₅)(R₃₆), or C(O)NH(R₃₇); each —R′and —R″ is independently a single bond, —H, -alkyl, -alkoxy,-cycloalkyl, -hydroxyalkyl, -aryl, or -heterocyclo, each of which isoptionally substituted, each —R₃ through —R₅, —R₇, —R₈, —R₂₁ through—R₂₃, and —R₂₅ through —R₂₇ is independently —H, -alkyl, -alkoxy,-halogen, -hydroxy, -cycloalkyl, -hydroxyalkyl, -aryl, or -heterocyclo,each of which is optionally substituted; each —R₁, —R₂, —R₆, R₉ through—R₁₂, —R₂₄, and —R₂₈ through —R₃₇ is independently —H, -alkyl, -alkenyl,-cycloalkyl, -aryl, or a 5- or 6-membered nitrogen or oxygen containingheterocycle; wherein —K₅ is a polydentate metal-chelating ligand radicalof formula V:

 wherein Q is the group—(C(RR))_(m1)—(Y¹)_(n)—(C(RR))_(m2)—(Y²—(C(RR))_(m3))_(n1); Y¹ and Y²are each independently —CH₂—, —NR—, —O—, —S—, —SO—, —SO₂— or —Se—; n andn1 are each independently 0 or 1; and m1, m2 and m3 are independently 0or an integer from 1 to 4; provided that m1 and m2 are not both 0, thatm1+m2+n+n1 is less than 6 and that a carbon atom bearing an R group isnot directly bonded to more than one heteroatom; each R and R* group isindependently; —H, —R₄; -alkoxy; -hydroxy; -halogen, -haloalkyl, —OR⁵,—C(O)—R⁵, —C(O)—N(R⁵)₂, —N(R⁵)₂, —N(R⁵)—COR⁵, -alkyl-C(O)—OR⁵,-alkyl-C(O)—N(R⁵)₂, -alkyl-N(R⁵)₂—, -alkyl-N(R⁵)—COR⁵, -aryl-C(O)—OR⁵,-aryl-C(O)—N(R⁵)₂—, -aryl-N(R⁵)₂—, -aryl-N(R⁵)—COR⁵, -nitrile, -acyl,-acyloxy, -heterocyclo, -hydroxyalkyl, -alkoxyalkyl, -hydroxyaryl,-arylalkyl, —SO₂—R⁵, -alkyl-SO₂—R⁵, or —[R³]—; wherein each —[R³]— is,in its entirety, the linking group —[(A)p*]— that serves to couple themetal chelating ligand radical —K₅ to —X—; each —R⁴ is independently —H,-alkyl, -alkoxy, -hydroxy, -cycloalkyl, -hydroxyalkyl, -aryl, or-heterocyclo, each of which is optionally substituted; each —R⁵ isindependently —H, -alkyl, -aryl, -cycloalkyl or -hydroxyalkyl, each ofwhich is independently substituted; or two R groups, or an R group andan R* group, taken together with the one or more atoms to which they arebonded, form a saturated or unsaturated, spiro or fused, carbocyclic orheterocyclic ring which is optionally substituted by one or more groupsR or R* groups above; each —G¹ and —G² is independently —OH or —(NR⁶)₂;with the proviso that at least one of —G¹ or —G² is —(NR⁶)₂, where each—R⁶ is independently -hydrogen, -alkyl, -aryl, -acyl or —[R³]—; and A isa linking group; and p is 0 or a positive integer; with the proviso thatat one to three —R , —R*, or —R⁶ groups is —[R³]—; or a pharmaceuticallyacceptable salt thereof.
 4. The composition of claim 3 wherein M₁ orboth M₁ and M₅ are paramagnetic or superparamagnetic metals and K₁ orboth —K₁ and —K₅ are enhanced relaxivity polyaza macrocyclic radicals offormula VI;

wherein n is 0 or 1; each m, o, and p is independently 1 or 2; Q is—[C(R′)(R″)]_(s1)—[C(t)(R₂₁)]_(s2)—[C(R₂₂)(R₂₃)]_(s3)—X3—Y—X4—; whereins1, s2, s3, and s4 are independently 0 to 2; Y is a single bond,—C(R₂₅)(R₂₆)—, or Y1 wherein, Y1 is —C(═X5)—X6—W—, wherein W is a singlebond, -alkylidene-, -cycloalkylidene-, -arylidene-, -alkenylidene-, or-alkynylidene-, whose carbon atoms are optionally substituted; t is H,R₂₇, —C(O)OR₂₈, —P(O)(OR₂₉))OH, —P(O)(OR₃₀))OR₃₁, —P(O)(OR₃₂)R₃₃,—P(O)(OH)R₃₄—C(O)N(R₃₅)(R₃₆), or C(O)NH(R₃₇); each G is independently—C(O)OR′″, —P(O)(OR′″)OH, —P(O)(OR′″)₂, —P(O)(OR′″)R″,—P(O)(OH)R″C(O)N(R′″)₂, or C(O)NH(R′″); each —R′ and —R″ isindependently a single bond, —H, -alkyl, -alkoxy, -cycloalkyl,hydroxyalkyl, -aryl, or -heterocyclo, each of which is optionallysubstituted, each —R′″ is independently a —H, -alkyl, -cycloalkyl,-hydroxyalkyl, -aryl, or -heterocyclo, each of which is optionallysubstituted, each —R₁₃ through —R₂₃, and —R₂₅ through —R₂₇ isindependently —H, -alkyl, -alkoxy, -halogen, -hydroxy, -cycloalkyl,-hydroxyalkyl, aryl, or -heterocyclo, each of which is optionallysubstituted; each —R₂₄, and —R₂₈ through —R₃₇ is independently —H,-alkyl, -alkenyl, -cycloalkyl, -aryl, a 5- or 6-membered nitrogen oroxygen containing heterocycle, each of which is optionally substituted;or R₁₃ together with R₁₅, and R₁₇ together with R₁₈, independently form,together with the carbon atoms in the polyazamacrocycle to which theyare attached, a fused fully or partially saturated non-aromaticcyclohexyl ring which are optionally substituted by one or more halogen,alkyl, ether, hydroxy, or hydroxyalkyl groups, and which are optionallyfused to a carbocyclic ring, or R₁₃ and R₁₅ are each hydrogen and R₁₇,together with R₁₈, forms a fused fully or partially saturatednon-aromatic cyclohexyl ring as defined above, or R₁₃, together withR₁₅, forms a fused fully or partially saturated non-aromatic cyclohexylring as defined above, and R₁₇ and R₁₈ are hydrogen; or apharmaceutically acceptable salt thereof.
 5. A conjugatable polyazamacrocyclic intermediate useful for the preparation of a composition forvisualization or radiotherapy of tissues or organs that overexpressfolate-binding protein using magnetic resonance imaging or neutroncapture therapy techniques comprising one or more folate-receptorbinding residues conjugated to one or more enhanced relaxivity polyazamacrocyclic radicals which are optionally chelated to a paramagnetic orsuperparamagnetic metal capable of either being detected outside thebody by imaging means for diagnosis or capable of providing aradiotherapeutic effect using neutron capture therapy; wherein saldfolate-receptor binding compound has the structure of formula IIc:

wherein R₀ is a folate-receptor binding moiety of formula:

each X is independently —O—, —S—, —NH—, or —NR₁—; n1 and n5 areindependently 0 or 1; b1 and b5 are independently 1 to 3; m1 and m5 areindependently 1 to 81; each —K₁ is independently —H, -alkyl, -alkenyl,-alkynyl, -alkoxy, -aryl, -alkyl, —CON(R₂)₂, -glutamate, -polyglutamate,or —K₄; each —K₂ is independently —H, -alkyl, -alkenyl, -alkynyl,-alkoxy, -aryl, -alkyl, —CON(R₂)₂, -glutamate, -polyglutamate, or —K₃;—K₃ is

M₁ and M₅ are paramagnetic or superparamagnetic metals; and —K₄ and —K₅are each independently enhanced-relaxivity polyaza macrocyclicmetal-chelating ligand radicals of formula VI that are optionallychelated to M₁ and M₅;

 wherein n is 0 or 1; each m, o, and p is independently 1 or 2; Q is—[C(R′)(R″)]_(s1)—[C(t)(R₂₁)]_(s2)—[C(R₂₂)(R₂₃)]_(s3)—X3—Y—X4—; whereins1, s2, s3, and s4 are independently 0 to 2; X3, X4, X5, and X6 areindependently a single bond, —O—, —S—, or —N(R₂₄)—; Y is a single bond,—C(R₂₅)(R₂₆)—, or Y1, wherein Y1 is —C(═X5)—X6—W—, wherein W is a singlebond, -alkylidene-, -cycloalkylidene-, -arylidene-, -alkenylidene-, or-alkynylidene-, whose carbon atoms are optionally substituted; t is H,R₂₇, —C(O)OR₂₈, —P(O)(OR₂₉))OH, —P(O)(OR₃₀))OR₃₁, —P(O)(OR₃₂)R₃₃,—P(O)(OH)R₃₄—C(O)N(R₃₅)(R₃₆), or C(O)NH(R₃₇); each G is independently—C(O)OR′″, —P(O)(OR′″)OH, —P(O)(OR′″)₂, —P(O)(OR′″)R″,—P(O)(OH)R″C(O)N(R′″)₂, or C(O)NH(R′″); each R′ and R″ is independentlya single bond, —H, -alkyl, -alkoxy, -cycloalkyl, -hydroxyalkyl, -aryl,or -heterocyclo, each of which is optionally substituted, each R′″ isindependently —H, -alkyl, -cycloalkyl, -hydroxyalkyl, -aryl, or-heterocyclo, each of which is optionally substituted, each —R₁₃ through—R₂₃, and —R₂₅ through —R₂₇ is independently —H, -alkyl, -alkoxy,-halogen, -hydroxy, -cycloalkyl, -hydroxyalkyl, -aryl, or -heterocyclo,each of which is optionally substituted; each —R₂₄, and —R₂₈ through—R₃₇ is independently —H, -alkyl, -alkenyl, -cycloalkyl, -aryl, a 5- or6-membered nitrogen or oxygen containing heterocycle, each of which isoptionally substituted; or R₁₃ together with R₁₅, and R₁₇ together withR₁₈, independently form, together with the carbon atoms in thepolyazamacrocycle to which they are attached, a fused fully or partiallysaturated non-aromatic cyclohexyl ring which may be unsubstituted orsubstituted by one or more halogen, alkyl, ether, hydroxy, orhydroxyalkyl groups, and which may be further fused to a carbocyclicring, or R₁₃ and R₁₅ are each hydrogen and R₁₇, together with R₁₈, formsa fused fully or partially saturated non-aromatic cyclohexyl ring asdefined above, or R₁₃, together with R₁₅, forms a fused fully orpartially saturated non-aromatic cyclohexyl ring as defined above, andR₁₇ and R₁₈ are hydrogen; —(A)p— and —(A)p*— are optional linkers eachindependently comprising a straight or branched chain made up ofmoieties that are the same or different and selected from the groupconsisting of; —CH₂—, —CHR₃—, —CR₄R₅—, —CH═CH—, —CH═CR₆—, >CR₇—CR₈<,—C═C—, —CR₉═CR₁₀—, —C≡C—, -cycloalkylidene-, -cycloalkenyl-,-arylidene-, -heterocyclo-, carbonyl (—CO—), —O—, —S—, —NH—, —HC═N—,—CR₁₁═N—, —NR₁₂—, —CS—,

 and p and p* are each individually 0 to 24; or —X—[(A)p]— or—X—[(A)p*]— in its entirety is the group —Q— as defined above each —R₃through —R₅, —R₇ and —R₈ is independently —H, -alkyl, -alkenyl, -alkoxy,-aryl, a 5- or 6-membered nitrogen or oxygen containing heterocycle,halogen, hydroxy or -hydroxyalkyl; and each —R₁, —R₂, —R₆, —R₉ through—R₁₂ is independently —H, -alkyl, -alkoxy, -cycloalkyl, -aryl,-heterocyclo, -hydroxy or -hydroxyalkyl; or a pharmaceuticallyacceptable salt thereof; said intermediate containing at least one freeamine, carboxylate or thiocarboxylate functionality that can be used forconjugation to targeting vectors such as folate, said intermediateshaving the structure of formula VIa:

wherein n is 0 or 1; each m, o, and p is independently 1 or 2; —Q(int)is a conjugatable amine-, carboxylate- or thiocarboxylate-containinggroup of formula —[C(R′)(R″)]s₁—[C(t)(R₂₁)]s₂—[C(R₂₂)(R₂₃)]s₃—X₃—Y—X₄;wherein s1, s2, s3, and s4 are independently 0 to 2; X₃ is a singlebond, —O—, —S—, —NH— or —NR₂₄— if Y is present, or X₃ is —OH, —SH, —NH₂or —N(R₂₄)H if Y and X₄ are absent; X₄ is a single bond, —OH, —COOH,—SH, —NHR₂₄ or —NH₂; Y is a single bond, —C(R₂₅)(R₂₆)—, or Y1 wherein,Y1 is —C(═X5)—X6—W—, wherein X5 is ═O or ═S; X₆ is a single bond, —SH,—NH(R₃₈), —NH₂ or —OH if W and X4 are absent, and is —S—, —O—, —NH—, or—N(R₃₉)—, if W and X₄ are present; W is a single bond, or is-alkylidene-, -cycloalkylidene-, -arylidene-, -alkenylidene-, or-alkynylidene-, whose carbon atoms are optionally substituted; t is —H,—R₂₇, —C(O)OR₂₈, —P(O)(OR₂₉))OH, —P(O)(OR₃₀))OR₃₁, —P(O)(OR₃₂)R₃₃,—P(O)(OH)R₃₄—C(O)N(R₃₅)(R₃₆), or —C(O)NH(R₃₇); each —G is independently—C(O)OR′″, —P(O)(OR′″)OH, —P(O)(OR′″)₂, —P(O)(OR′″)R″,—P(O)(OH)R″—C(O)N(R′″)₂, or —C(O)NH(R′″); each —R′ and —R″ isindependently a single bond, —H, -alkyl, -alkoxy, -cycloalkyl,-hydroxyalkyl, -aryl, or -heterocyclo, each of which is optionallysubstituted, each —R′″ is independently —H, -alkyl, -cycloalkyl,-hydroxyalkyl, -aryl, or -heterocyclo, each of which is optionallysubstituted, each —R₁₃ through —R₂₃, —R₂₅ through —R₂₇ is independently—H, -alkyl, alkoxy, -halogen, -hydroxy, -cycloalkyl, -hydroxyalkyl,-aryl, or -heterocyclo, each of which is optionally substituted; each—R₂₄, and —R₂₈ through —R₃₉ is independently —H, -alkyl, -alkenyl,cycloalkyl, aryl, a 5- or 6-membered nitrogen or oxygen containingheterocycle, each of which is optionally substituted; or R₁₃ togetherwith R₁₅, and R₁₇ together with R₁₈, independently form, together withthe carbon atoms in the polyazamacrocycle to which they are attached, afused fully or partially saturated non-aromatic cyclohexyl ring whichare optionally substituted by one or more halogen, alkyl, ether,hydroxy, or hydroxyalkyl groups, and which are optionally further fusedto a carbocyclic ring, or R₁₃ and R₁₅ are each hydrogen and R₁₇,together with R₁₈, forms a fused full or partially saturatednon-aromatic cyclohexyl ring as defined above, or R₁₃, together withR₁₅, forms a fused fully or partially saturated non-aromatic cyclohexylring as defined above, and R₁₇ and R₁₈ are hydrogen; or apharmaceutically acceptable thereof.
 6. A composition comprisingfolate-receptor binding ligands and a pharmaceutically acceptablecarrier for use in nuclear medicine, magnetic resonance imaging, orneutron capture therapy techniques, said folate-receptor binding ligandscomprising dendrimeric first-, second-, third-, and fourth-generationconjugates containing one folate-receptor binding moiety coupled to oneor more macrocyclic metal-chelating ligand radicals that are optionallychelated to paramagnetic, superparamagnetic, radioactive ornon-radioactive metals for detection outside the body by imaging meansfor diagnosis or for providing a therapeutic or radiotherapeutic effect;wherein said folate-receptor binding compounds have the structure offormulae VIIa-VIId:

wherein R₀ is a folate-receptor moiety of formula:

wherein for the first generation dendrimers of formula VIIa, bearing onefolate-receptor binding moiety and 3 or 6 metal chelating ligandradicals: W₁ and W₂ are each independently —OR′″, —SR′″,—NR′″R′″—CON(R₂)₂, -glutamate, -polyglutamate, or —K₆; wherein each —R′″is independently -cycloalkyl, -hydroxyalkyl, or -heterocyclo; with theproviso that either W₁, W₂, or both W₁ and W₂ of formula VIIa must be—K₆, where —K₆ is a moiety of formula VIIIa;

 wherein Y is —Y′—C(═X)— wherein X is ═O or ═S; Y′ is N(R₆)—Z—; whereinZ is a single bond, -alkylidene-, -vinylidene-, -cycloalkylidene-, or-arylidene-; A is —C(═O)—, C(═S), or —CH₂—N(R₇)—; M₁ is asuperparamagnetic, paramagnetic, radioactive or non-radioactive metal,and K₁ is a macrocyclic metal chelating ligand moiety; and, wherein forsecond generation dendrimers, bearing one folate receptor binding moietyand 9 or 18 macrocyclic metal-chelating ligand radicals and having thestructure of formula VIIb: W₁ and W₂ are each independently —OR′″,—SR′″—NR′″R′″, or —K₇, wherein each —R′″ is independently —H, -alkyl,-aryl, -cycloalkyl, -hydroxyalkyl, or -heterocyclo, and —K₇ is a residueof formula VIIIb; with the proviso that either W₁, W₂, or both W₁ and W₂must be —K₇

wherein Y is a single bond or —Y′—C(═X)— wherein X is ═O or ═S and Y′ is—N(R₆)—Z—; wherein Z is a single bond, -alkylidene-, -vinylidene-,-cycloalkylidene-, or -arylidene-; A is —C(O)—, C(S)—, or —CH₂—N(R₇)—; Dis —N(R₆)—C— if A is —C(O)— or —C(S)— or —C(═X₂)—E—N(R₇)—C— if A is—CH₂—N(R₇)—; wherein E is a single bond, -alkylidene-, -vinylidene-,-cycloalkylidene-, or -arylidene- and X₂ is ═O or ═S; and wherein forthe third generation dendrimeric compounds of formula VIIc; bearing onefolate receptor binding residue and 27 or 54 macrocyclic metal-chelatingligand radicals: W₁ and W₂ are each independently —OR′″, —SR′″,—NR′″R′″, or —K₈ wherein each —R′″ is independently —H, -alkyl, -aryl,-cycloalkyl, -hydroxyalkyl, or -heterocyclo, and —K₈ is a moiety offormula VIIIc; with the proviso that either W₁, W₂, or both W₁ and W₂ ofthe compounds of formula VIIc must be —K₈:

 wherein, Y is a single bond or —Y′—C(═X)— wherein X is ═O or ═S; Y′ is—N(R₆)—Z—; wherein Z is a single bond, -alkylidene-, -vinylidene-,-cycloalkylidene-, or -arylidene-; A is —C(O)—, —C(S)—, or —CH₂—N(R₇)—;D₁ and D₂ are each independently —N(R₆)—C if A is —C(O)— or —C(S)—, and—C(═X₂)—E—N(R₇)—C if A is —CH₂—N(R₇)—; wherein E is a single bond,-alkylidene-, -vinylidene-, -cycloalkylidene-, or -arylidene- and X₂ is═O or ═S; and wherein for the fourth generation dendrimeric compounds offormula VIId; bearing one folate receptor binding moiety and 81 or 162macrocyclic metal-chelating ligand radicals; W₁ and W₂ are eachindependently —OR′″, —SR′″, —NR′″R′″ or —K₉, wherein each R′″ isindependently —H, -alkyl, -aryl, -cycloalkyl, -hydroxyalkyl, or-heterocyclo and —K₉ is a moiety of formula VIIId; with the proviso thateither W₁, W₂, or both W₁ and W₂ of the compounds of formula VIId mustbe —K₉);

 wherein Y is a single bond or —y′—C(═X)— wherein X is ═O or ═S; Y′ is—N(R₆)—Z—; wherein Z is a single bond, -alkylidene-, -vinylidene-,-cycloalkylidene-, or -arylidene-; A is —C(O)—, —C(S)—, or —CH₂—N(R₇)—;D₁, D₂, and D₃ are each independently —N(R₆)—C if A is —C(O)— or C(S)—,and —C(═X₂)—E—N(R₇)—C if A is —CH₂—N(R₇)—; wherein E is a single bond,-alkylidene-, -vinylidene-, -cycloalkylidene-, or -arylidene- and X₂ is═O or ═S; and each —R₁ to —R₇ of the compounds of formula VIIIa-VIIId isindependently —H, -alkyl, -hydroxyalkyl, -alkoxy, -alkoxyalkyl,-cycloalkyl, or -aryl; each of which is optionally substituted, or apharmaceutically acceptable salt thereof.
 7. The composition of claim 6wherein W₁ of formula VIIa-VIId is a moiety of formula VIIIa, VIIIb,VIIIc or VIIId and W₂ of formula VIIa-VIId is —OR′″, —SR′″,—NR′″R′″—CON(R₂)₂, -glutamate, or -polyglutamate, wherein each R′″ isindependently -aryl, -cycloalkyl, or -heterocyclo.
 8. The composition ofclaim 6 wherein W₂ of formula VIIa-VIId is a moiety of formula VIIIa ,VIIIb, VIIIc or VIIId; and W₁ of formula VIIa-VIId is —OR′″, —SR′″,—NR′″R′″—CON(R₂)₂, -glutamate, or -polyglutamate, wherein each R′″ isindependently aryl, -cycloalkyl, or -heterocyclo.
 9. The composition ofclaim 6 wherein both W₁ and W₂ of formula VIIa-VIId is a moiety offormula VIIIb, VIIIc or VIIId].
 10. The composition of formula VIIa-VIIdof claim 6 wherein M₁ is a radioactive-, paramagnetic- orsuperparamagnetic- metal and each K₁ is a macrocyclic metal chelatingligand radical of formula VI;

wherein said metal chelating radical is attached to the remainder of thecompound of formulae VIIa-VIId via the free —N(R)— atom of the function—Q— if A is —C(O)— or —C(S)— or through the free —C(O)— atom of thefunction —Q— if A is —CH₂—N(R₇)—; wherein —Q— is—[C(R′)(R″)]_(s1)—[C(t)(R₂₁)]_(s2)—[C(R₂₂)(R₂₃)]_(s3)—X3—Y—X4—; whereins1, s2, s3, and s4 are independently 0 to 2; X3, X4, X5, and X6 areindependently a single bond, —O—, —S—, or —N(R₂₄)—; Y is a single bond,—C(R₂₅)(R₂₆)—, or Y1, wherein Y1 is —C(═X5)—X6—W—, wherein W is a singlebond, -alkylidene-, -cycloalkylidene-, -arylidene-, -alkenylidene-, or-alkynylidene-, whose carbon atoms are optionally substituted; t is H,R₂₇, —C(O)OR₂₈, —P(O)(OR₂₉))OH, —P(O)(OR₃₀))OR₃₁, —P(O)(OR₃₂)R₃₃,—P(O)(OH)R₃₄, —C(O)N(R₃₅)(R₃₆), or C(O)NH(R₃₇); each G is independently—C(O)OR′″, —P(O)(OR′″)OH, —P(O)(OR′″)₂, —P(O)(OR′″)R″,—P(O)(OH)R″C(O)N(R′″)₂, or C(O)NH(R′″); each R′ and R″ is independentlya single bond, —H, -alkyl, -alkoxy, -cycloalkyl, -hydroxyalkyl, -aryl,or -heterocyclo, each of which is optionally substituted, each R′″ isindependently —H, -alkyl, -cycloalkyl, -hydroxyalkyl, -aryl, or-heterocyclo, each of which is optionally substituted, each —R₁₃ through—R₂₃, and —R₂₅ through —R₂₇ is independently —H, -alkyl, -alkoxy,-halogen, -hydroxy, -cycloalkyl, -hydroxyalkyl, -aryl, or -heterocyclo,each of which is optionally substituted; each —R₂₄, and —R₂₈ through—R₃₇ is independently —H, -alkyl, -alkenyl, -cycloalkyl, -aryl, or a 5-or 6-membered nitrogen or oxygen containing heterocycle, each of whichis optionally substituted; or a pharmaceutically accepted salt thereof.11. The composition of formula VIIa-VIId of claim 6 wherein M₁ is aradioactive metal and at least one —K₁ is a macrocyclic metal chelatingligand radical of formula V:

wherein —Q— is the group—(C(RR))_(m1)—(Y¹)_(n)—(C(RR))_(m2)—(Y²—(C(RR))_(m3))_(n1); Y¹ and Y²are each independently —CH₂—, —NR—, —O—, —S—, —SO—, —SO₂— or —Se—; n andn1 are each independently 0 or 1; and m1, m2 and m3 are independently 0or an integer from 1 to 4; provided that m1 and m2 are not both 0, thatm1+m2+n+n1 is less than 6 and that a carbon atom bearing an R group isnot directly bonded to more than one heteroatom; each —R and —R* groupis independently: —R⁴; -alkoxy; -hydroxy; -halogen, especially fluoro,-haloalkyl, —OR⁵, —C(O)—R⁵, —C(O)—N(R⁵)₂, —N(R⁵)₂, —N(R⁵)—COR⁵,-alkyl-C(O)—OR⁵, -alkyl-C(O)—N(R⁵)₂, -alkyl-N(R⁵)₂—, -alkyl-N(R⁵)—COR⁵,-aryl-C(O)—OR⁵, -aryl-C(O)—N(R⁵)₂, aryl-N(R⁵)₂—, -aryl-N(R⁵)—COR⁵,-nitrile, -acyl, -acyloxy, -heterocyclo, -hydroxyalkyl, -alkoxyalkyl,-hydroxyaryl, arylalkyl, —SO₂—R⁵, -alkyl-SO₂—R⁵, or —[R³]—; wherein—[R³]— is a linking group —[(A)p]— that links the metal chelating ligandradical of formula V to the remainder of the molecule of formulae VIIathrough VIId; wherein —[(A)p]— comprises a straight or branched chain ofindividual moieties that are the same or different and selected from thegroup consisting of: —CH₂—, —CHR₃—, —CR₄R₅—, —CH═CH—, —CH═CR₆—,>CR₇—CR₈<, —C═C—, —CR₉═CR₁₀—, —C≡C—, -cycloalkylidene-, -cycloalkenyl-,-arylidene-, -heterocyclo-, carbonyl (—CO—), —O—, —S—, —NH—, —HC═N—,—CR₁₁═N—, —NR₁₂—, (—CS—),

and p is an integer from 0 to 24; each —R⁴ and —R³ through —R⁵ isindependently —H, -alkyl, -alkoxy, -hydroxy, -cycloalkyl, -hydroxyalkyl,-aryl, or -heterocyclo, each of which is optionally substituted; each—R⁵ and —R⁶ through —R¹² is independently —H, -alkyl, -aryl, -cycloalkylor -hydroxyalkyl, each of which is independently substituted; or two Rgroups, or an R group and an R* group, taken together with the one ormore atoms to which they are bonded, form a saturated or unsaturated,spiro or fused, carbocyclic or heterocyclic ring which is optionallysubstituted by one or more R or R* groups; each —G¹ and —G² isindependently —OH or —(NR⁶)₂; with the proviso that at least one of —G¹or —G² is —(NR⁶)₂, and each —R⁶ is independently -hydrogen, -alkyl,-aryl, -acyl or —[R³]—; with the proviso that at least one —R , —R*, or—R⁶ group is —[R³]—; or a pharmaceutically acceptable salt thereof. 12.The composition of formula VIIa-VIId of claim 6 wherein M₁ is aradioactive isotope and at least one K₁ is a macrocyclic metal chelatingligand of formula IIIa-IIIc:

wherein Q is the group—(C(RR))_(m1)—Y¹(C(RR))_(m2)—(Y²(C(RR))_(m3))_(n)—, wherein Y¹ and Y²are independently —CH₂—, —NR—, —O—, —S—, —SO—, —SO₂— or —Se—; n is 0 or1; and m1, m2 and m3 are integers from 0 to 4, provided that the sum ofm1 and m2 is greater than zero; all R and R* groups are independently—R⁴, —Cl, —F, —Br, —OR⁵, —COOR⁵, —CON(R⁵)₂, —N(R⁵)₂, -alkyl-COOR⁵,-alkyl-C(O)—N(R⁵)₂, -alkyl-N(R⁵)₂, —C(O)OR⁵, —C(O)N(R⁵)₂, -aryl-N(R⁵)₂,-acyl, -acyloxy, -heterocyclo, -hydroxyalkyl, —SO₂—R⁵, -alkyl-SO₂—R⁵, or—[R³]—; wherein —[R³]— is a linking group —[(A)p]— that links the metalchelating ligand of formula IIIa, IIIb, or IIIc to the remainder of themolecule; wherein —[(A)p]— comprises a straight or branched chain ofindividual moieties that are the same or different and selected from thegroup consisting of: —CH₂—, —CHR₃—, —CR₄R₅—, —CH═CH—, —CH═CR₆—,>CR₇—CR₈<, —C═C—, —CR₉═CR₁₀—, —C≡C—, -cycloalkylidene-, -cycloalkenyl-,-arylidene-, -heterocyclo-, carbonyl —(CO)—, —O—, —S—, —NH—, —HC═N—,—CR₁₁═N—, —NR₁₂—, —(CS)—

 and p is an integer from 0 to 24; each —R⁴ and —R₃ through —R₅ isindependently —H, -alkyl, -alkoxy, -hydroxy, -cycloalkyl, -hydroxyalkyl,-aryl, or -heterocyclo, each of which is optionally substituted; each—R⁵ and —R₆ through —R₁₂ is independently —H, -alkyl, -aryl, -cycloalkylor -hydroxyalkyl, each of which is independently substituted; with theprovisos that a carbon atom bearing an —R group is not directly bondedto more than one heteroatom; and that at least one —R or —R* group on—K₁ is —[R³]— or a pharmaceutically acceptable salt thereof.
 13. Afolate-receptor binding ligand comprising dendrimeric first-, second-,third-, and fourth- generation conjugates containing one or morefolate-receptor binding moieties coupled to one or more macrocyclicmetal-chelating ligand radicals for detection outside the body byimaging means for diagnosis or for providing a therapeutic orradiotherapeutic effect, wherein said folate-receptor binding ligandshave the structure of formulae IXa, IXb, IXc, and IXd, representingdendrimers of generations 1, 2, 3, and 4, respectively, wherein for thefirst generation dendrimers of formula IXa, bearing three folate andthree metal chelating ligand radicals;

F is a folate-receptor binding moiety of formula:

 wherein R₀ is a moiety of formula:

each X₁ through X₄ is independently ═O or ═S; each A is —C(O)—, —C(S)—,or —CH₂—N(R₇)—; E is a single bond, -alkylidene-, -vinylidene-,-cycloalkylidene-, or -arylidene-; B is a macrocyclic metal-chelatingligand radical that is attached to A via an amide or thioamide bond andis optionally chelated to a paramagnetic, superparamagnetic, radioactiveor non-radioactive metal; —R₁, —R₆ through —R₈, —R₁₃, and —R₁₄ areindependently —H, -alkyl, -hydroxyalkyl, -cycloalkyl, or -aryl; —R₂through —R₅ and —R₉ through —R₁₂ are independently —H, -alkyl,-hydroxyalkyl, -alkoxy, -hydroxyalkyl, -halogen, -cycloalkyl, -aryl or-heterocyclo; or a pharmaceutically accepted salt thereof; and whereinfor the second generation dendrimeric compounds of formula IXb, bearingnine folate-receptor binding moieties and nine metal-chelating ligandradicals:

A, B, E, F, X₁ through X₄ and all —R groups are as defined for thecompounds of formula IXa; D₁ and D₂ are independently —N(R₆)—C if A is—C(O)— or —C(S)—, and —C(═X₃)—E—N(R₇)—C if A is —CH₂—N(R₇)—; and whereinfor the third generation dendrimeric compounds of formula IXc, bearing27 folate receptor binding moieties and 27 metal chelating ligandradicals:

D₁, D₂, D₃, and D₄ are independently —N(R₆)—C if A is —C(O)— or —C(S)—,and —C(═X₃)—E—N(R₇)—C if A is —CH₂—N(R₇)—; and all other groups aredefined as above; and wherein for the fourth generation dendrimericcompounds of formula IXd, bearing 81 folate receptor binding moieties to81 metal chelating ligands:

D₁, D₂, D₃, D₄, D₅, and D₆ are each independently —N(R₆)—C if A is—C(O)— or —C(S)—, and —C(═X₃)—E—N(R7)—C if A is —CH₂—N(R₇)—; or apharmaceutically acceptable salt thereof.
 14. The folate-receptorbinding ligand of claim 13 wherein F of formulae IXb, IXc, and IXd is afolate-receptor binding moiety of formula:

wherein R₀ is a moiety of formula:

or a pharmaceutically acceptable salt thereof.
 15. The folate-receptorbinding ligand of claim 13 wherein F of formulae IXb, IXc, and IXd is afolate receptor binding moiety of formula:

wherein R₀ is a moiety of formula:

or a pharmaceutically acceptable salt thereof.
 16. The folate receptorbinding ligand of formulae IXb, IXc, and IXd of claim 13, wherein B is apolyaza macrocyclic ligand radical of formula VIc that is optionallychelated to a paramagnetic, superparamagnetic, radioactive ornon-radioactive metal,

wherein said macrocyclic ligand radical is attached to A via an amide orthioamide linkage through a free—C(O)— group of the function —Q— if A is—CH₂—N(R₇)—; —Q— is—[C(R′)(R″)]_(s1)—C(t)(R₂₁)]_(s2)—[C(R₂₂)(R₂₃)]_(s3)—X₃—Y—X₄—; whereins1, s2, s3, and s4 are independently 0 to 2; —X₃, —X₄, —X₅, and —X₆ areindependently a single bond, —O—, —S—, or —N(R₂₄)—; Y is a single bond,—C(R₂₅)(R₂₆)—, or Y1, wherein Y1 is —C(═X₅)—X₆—W—, wherein W is a singlebond, -alkylidene-, -cycloalkylidene-, arylidene-, -alkenylidene-, or-alkynylidene-, whose carbon atoms are optionally substituted; t is H,R₂₇, —C(O)OR₂₈, —P(O)(OR₂₉))OH, —P(O)(OR₃₀))OR₃₁, —P(O)(OR₃₂)R₃₃,—P(O)(OH)R₃₄, —C(O)N(R₃₅)(R₃₆), or C(O)NH(R₃₇); each G is independently—C(O)OR′″, —P(O)(OR′″)OH, —P(O)(OR′″)₂, —P(O)(OR′″)R″,—P(O)(OH)R″—C(O)N(R′″)₂, or —C(O)NH(R′″); each —R′ and —R″ isindependently a single bond, —H, -alkyl, -alkoxy, -cycloalkyl,-hydroxyalkyl, -aryl, or -heterocyclo, each of which is optionallysubstituted, each —R′″ is independently —H, -alkyl, -cycloalkyl,-hydroxyalkyl, -aryl, or -heterocyclo, each of which is optionallysubstituted, each —R_(13c) through —R_(20c), —R₂₁ through —R₂₃, and —R₂₅through —R₂₇ is independently —H, -alkyl, -alkoxy, -halogen, -hydroxy,-cycloalkyl, -hydroxyalkyl, -aryl, or -heterocyclo, each of which isoptionally substituted; each —R₂₄, and —R₂₈ through —R₃₇ isindependently —H, -alkyl, -alkenyl, -cycloalkyl, -aryl, a 5- or6-membered nitrogen or oxygen-containing heterocycle, each of which isoptionally substituted; or a pharmaceutically accepted salt thereof. 17.The folate-receptor binding ligand of formulae IXa, IXb, IXc, and IXd ofclaim 13 wherein B is a metal-chelating ligand radical of formulaIIIa-IIIc that is optionally chelated to a paramagnetic,superparamagnetic, radioactive or non-radioactive metal:

wherein Q is the group—(C(RR))_(m1)—Y¹(C(RR))_(m2)—(Y²—(C(RR))_(m3))_(n)—, wherein Y¹ and Y²are independently —CH₂—, —NR—, —O—, —S—, —SO—, —SO₂— or —Se—; n is 0 or1; and m1, m2 and m3 are integers from 0 to 4, provided that the sum ofm1 and m2 is greater than zero; all R and R* groups are independently—R⁴, —Cl, —F, —Br, —OR⁵, —COOR⁵, —CON(R⁵)₂, —N(R⁵)₂, -alkyl-COOR⁵,-alkyl-C(O)—N(R⁵)₂, -alkyl-N(R⁵)₂, —C(O)OR⁵, —C(O)N(R⁵)₂, -aryl-N(R⁵)₂,acyl, acyloxy, heterocyclo, hydroxyalkyl, —SO₂—R⁵, -alkyl-SO₂—R⁵, or—[R³]—; wherein —[R³]— is a linking group —[(A)p]— that couples themetal chelating radical of formula IIIa, IIIb, or IIIc to the remainderof the molecule; —[(A)p]— comprises a straight or branched chain ofindividual moieties that are the same or different and selected from thegroup consisting of; —CH₂—, —CHR₃—, —CR₄R₅—, —CH═CH—, —CH═CR₆—,>CR₇—CR₈<, —C═C—, —CR₉═CR₁₀—, —C≡C—, -cycloalkylidene-, -cycloalkenyl-,-arylidene-, -heterocyclo-, carbonyl —(CO)—, —O—, —S—, —NH—, —HC═N—,—CR₁₁═N—, —NR₁₂—, —CS—, and

 and p is an integer from 0 to 24; each —R⁴ and —R₃ through —R₅ isindependently —H, -alkyl, -alkoxy, -hydroxy, -cycloalkyl, -hydroxyalkyl,-aryl, or -heterocyclo, each of which is optionally substituted; each—R⁵ and —R₆ through —R₁₂ is independently —H, -alkyl, -aryl, -cycloalkylor -hydroxyalkyl, each of which is independently substituted; with theprovisos that a carbon atom bearing an —R group is not directly bondedto more than one heteroatom; and that at least one —R or —R* group onthe metal chelating radical —K₁ of formulae IIIa, IIIb, or IIIc is—[R³]—; or a pharmaceutically acceptable salt thereof.
 18. Thefolate-receptor binding ligand of formulae IXa, IXb, IXc, and IXd ofclaim 13, wherein B is a metal-chelating ligand radical of formula Vthat is optionally chelated to a paramagnetic, superparamagnetic,radioactive or non-radioactive metal:

wherein —Q— is the group—(C(RR))_(m1)—(Y¹)_(n)—(C(RR))_(m2)—(Y²—(C(RR))_(m3))_(n1); Y¹ and Y²are each independently —CH₂—, —NR—, —O—, —S—, —SO—, —SO₂— or —Se—; n andn1 are each independently 0 or 1; and m1, m2 and m3 are 0 or an integerfrom 1 to 4; provided that m1 and m2 are not both 0, that m1+m2+n+n1 isless than 6 and that a carbon atom bearing an R group is not directlybonded to more than one heteroatom; each —R and —R* group isindependently: —R⁴; -alkoxy; -hydroxy; -halogen, -haloalkyl, —OR⁵,—C(O)—R⁵, —C(O)—N(R⁵)₂, —N(R⁵)₂, —N(R⁵)—COR⁵, -alkyl-C(O)—OR⁵,-alkyl-C(O)—N(R⁵)₂, -alkyl-N(R⁵)₂—, -alkyl-N(R⁵)—COR⁵, -aryl-C(O)—OR⁵,-aryl-C(O)—N(R⁵)₂, aryl-N(R⁵)₂—, -aryl-N(R⁵)—COR⁵, -nitrile, -acyl,-acyloxy, -heterocyclo, -hydroxyalkyl, -alkoxyalkyl, -hydroxyaryl,arylalkyl, —SO₂—R⁵, -alkyl-SO₂—R⁵, or —[R³]—; wherein —[R³]— is alinking group —[(A)p]— that links the metal chelating ligand radical offormula V to the remainder of the molecule of formulae IXa, IXb, IXc,and IXd; wherein —[(A)p]— comprises a straight or branched chain ofindividual moieties that are the same or different and are selected fromthe group consisting of: —CH₂—, —CHR₃—, —CR₄R₅—, —CH═CH—, —CH═CR₆—,>CR₇—CR₈<, —C═C—, —CR₉═CR₁₀—, —C≡C—, -cycloalkylidene-, -cycloalkenyl-,-arylidene-, -heterocyclo-, carbonyl (—CO—), —O—, —S—, —NH—, —HC═N—,—CR₁₁═N—, —NR₁₂—, —CS—),

 and p is an integer from 0 to 24; each —R⁴ and —R₃ through —R₅ isindependently —H, -alkyl, -alkoxy, -hydroxy, -cycloalkyl, -hydroxyalkyl,-aryl, or -heterocyclo, each of which is optionally substituted; each—R⁵ and —R₆ through —R₁₂ is independently —H, -alkyl, -aryl, -cycloalkylor -hydroxyalkyl, each of which is independently substituted; or two Rgroups, or an R group and an R* group, taken together with the one ormore atoms to which they are bonded, form a saturated or unsaturated,spiro or fused, carbocyclic or heterocyclic ring which may beunsubstituted or substituted by one or more groups of R or R*; each —G¹and —G² is independently —OH or —(NR⁶)₂; with the proviso that at leastone of —G¹ or —G² is —(NR⁶)₂, and each —R⁶ is independently -hydrogen,-alkyl, -aryl, -acyl or —[R³]—; with the provisos that a carbon atombearing an —R group is not directly bonded to more than one heteroatomand that at least one —R , —R*, or —R⁶ group on the metal chelatingradical —K₁ of formula V is —[R³]—; or a pharmaceutically acceptablesalt thereof.
 19. The composition of claim 6 wherein W₁, W₂ or both W₁and W₂ contain metal chelating ligands of formula V that are chelated toa radioactive metal:

wherein Q is the group—(C(RR))_(m1)—(Y¹)_(n)—(C(RR))_(m2)—(Y²—(C(RR))_(m3))_(n1); Y¹ and Y²are each independently —CH₂—, —NR—, —O—, —S—, —SO—, —SO₂— or —Se—; n andn1 are each independently 0 or 1; and m1, m2 and m3 are independently 0or an integer from 1 to 4; provided that m1 and m2 are not both 0, thatm1+m2+n+n1 is less than 6 and that a carbon atom bearing an R group isnot directly bonded to more than one heteroatom; each R and R* group isindependently: —H, —R⁴; -alkoxy; -hydroxy; -halogen, especially fluoro,-haloalkyl, —OR⁵, —C(O)—R⁵, —C(O)—N(R⁵)₂, —N(R⁵)₂, —N(R⁵)—COR⁵,-alkyl-C(O)—OR⁵, -alkyl-C(O)—N(R⁵)₂, -alkyl-N(R⁵)₂—, -alkyl-N(R⁵)—COR⁵,-aryl-C(O)—OR⁵, -aryl-C(O)—N(R⁵)₂, aryl-N(R⁵)₂—, -aryl-N(R⁵)—COR⁵,-nitrile, -acyl, -acyloxy, -heterocyclo, -hydroxyalkyl, -alkoxyalkyl,-hydroxyaryl, -arylalkyl, —SO₂—R⁵, -alkyl-SO₂—R⁵, or —[R³]—; whereineach —[R³]— is, in its entirety, the linking group —[(A)p*]— that servesto couple the metal chelating ligand radical —K₅ to —X—; each —R⁴ isindependently —H, -alkyl, -alkoxy, -hydroxy, -cycloalkyl, -hydroxyalkyl,-aryl, or -heterocyclo, each of which is optionally substituted; each—R⁵ is independently —H, -alkyl, -aryl, -cycloalkyl or -hydroxyalkyl,each of which is independently substituted; or two R groups, or an Rgroup and an R* group, taken together with the one or more atoms towhich they are bonded, form a saturated or unsaturated, spiro or fused,carbocyclic (such as fused 1,2-phenyl) or heterocyclic ring which may beunsubstituted or substituted by one or more groups R or R* groups above;each —G¹ and —G² is independently —OH or —(NR⁶)₂; with the proviso thatat least one of —G¹ or —G² is —(NR⁶)₂, where each —R⁶ is independently-hydrogen, -alkyl, -aryl, -acyl or —[R³]—; and A is a linking group; andp is 0 or a positive integer; with the proviso that at one to three —R,—R*, or —R⁶ groups is —[R³]—; or a pharmaceutically acceptable saltthereof.
 20. The composition of claim 6 wherein W₁, W₂ or both W₁ and W₂contain metal chelating ligands of formula V that are chelated to aradioactive metal:

wherein n is 0; each m, o, and p is independently 1 or 2; Q is—[C(R′)(R″)]_(s1)—[C(t)(R₂₁)]_(s2)——[C(R₂₂)(R₂₃)]_(s3)—X3—Y—X4—; whereins1, s2, s3, and s4 are independently 0 to 2; X3, X4, X5 and X6 areindependently a single bond, —O—, —S—, or —N(R₂₄)—; Y is a—C(R₂₅)(R₂₆)—, or Y1 wherein Y1 is —C(═X5)—X6—W—, wherein W is a singlebond, -alkylidene-, -cycloalkylidene-, -arylidene-, -alkenylidene-, or-alkynylidene-, whose carbon atoms may or may not be substituted; t isH, R₂₇, —C(O)OR₂₈, —P(O)(OR₂₉))OH, —P(O)(OR₃₀))OR₃₁, —P(O)(OR₃₂)R₃₃,—P(O)(OH)R₃₄—C(O)N(R³⁵)(R₃₆), or C(O)NH(R₃₇); each G is independently—C(O)OR′″, —P(O)(OR′″)OH, —P(O)(OR′″)₂, —P(O)(OR′″)R″,—P(O)(OH)R″C(O)N(R′″)₂, or C(O)NH(R′″); each —R′ and —R″ isindependently a single bond, —H, -alkyl, -alkoxy, -cycloalkyl,hydroxyalkyl, -aryl, or -heterocyclo, each of which is optionallysubstituted, each —R′″ is independently a —H, -alkyl, -cycloalkyl,-hydroxyalkyl, -aryl, or -heterocyclo, each of which is optionallysubstituted; each —R₁₃ through —R₂₃, and —R₂₄, through —R₂₇ isindependently —H, -alkyl, -alkoxy, -halogen, -hydroxy, -cycloalkyl,-hydroxyalkyl, aryl, or -heterocyclo, each of which is optionallysubstituted; each —R₂₄, and —R₂₈ through —R₃₇ is independently —H,-alkyl, -alkenyl, -cycloalkyl, -aryl, a 5- or 6-membered nitrogen oroxygen containing heterocycle, each of which is optionally substituted;or R₁₃ together with R₁₅, and R₁₇ together with R₁₈, independently form,together with the carbon atoms in the poly-aza macrocycle to which theyare attached, a fused fully or partially saturated non-aromaticcyclohexyl ring which may be unsubstituted or substituted by one or morehalogen, alkyl, ether, hydroxy, or hydroxyalkyl groups, and which may befurther fused to a carbocyclic ring, or R₁₃ and R₁₅ are each hydrogenand R₁₇, together with R₁₈, forms a fused fully or partially saturatednon-aromatic cyclohexyl ring as defined above, or R₁₃, together withR₁₅, forms a fused fully or partially saturated non-aromatic cyclohexylring and R₁₇ and R₁₈ are hydrogen; or a pharmaceutically acceptable saltthereof.